Interleukin-9 mutein peptides

ABSTRACT

A C to T DNA variation at position 3365 in exon 5 of the human Asthma Associated Factor 1 (AAF1) produces the predicted amino acid substitution of a methionine for a threonine at codon 117 of AAF1. When this substitution occurs in both alleles in one individual, it is associated with less evidence of atopic allergy including asthma, fewer abnormal skin test responses, and a lower serum total IgE. Thus, applicant has identified the existence of a non-asthmatic, non-atopic phenotype characterized by methionine at codon 117 when it occurs in both AAF1 gene products in one individual.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. Provisional Application Ser. No.60/002,765 which was filed Aug. 24, 1995.

FIELD OF THE INVENTION

This invention relates to regulating IL-9 activity and treating atopicallergies and related disorders like asthma, based upon the relationshipbetween IL-9 and its receptor.

BACKGROUND OF THE INVENTION

Inflammation is a complex process in which the body's defense systemcombats foreign entities. While the battle against foreign entities maybe necessary for the body's survival, some defense systems improperlyrespond to foreign entities, even innocuous ones, as dangerous andthereby damage surrounding tissue in the ensuing battle.

Atopic allergy is an ecogenetic disorder, where genetic backgrounddictates the response to environmental stimuli. The disorder isgenerally characterized by an increased ability of lymphocytes toproduce IgE antibodies in response to ubiquitous antigens. Activation ofthe immune system by these antigens leads to allergic inflammation andmay occur after ingestion, penetration through the skin, or afterinhalation. When this immune activation occurs and pulmonaryinflammation ensues this disorder is broadly characterized as asthma.Certain cells are critical to this inflammatory reaction and theyinclude T cells and antigen presenting cells, B cells that produce IgE,and mast cells/basophils and eosinophils that bind IgE. Theseinflammatory cells accumulate at the site of allergic inflammation andthe toxic products they release contribute to the tissue destructionrelated to the disorder.

While asthma is generally defined as an inflammatory disorder of theairways, clinical symptoms arise from intermittent air flow obstruction.It is a chronic disabling disorder that appears to be increasing inprevalence and severity¹. It is estimated that 30-40% of the populationsuffer with atopic allergy, and 15% of children and 5% of adults in thepopulation suffer from asthma.¹ Thus, an enormous burden is placed onour health care resources.

The mechanism of susceptibility to atopy and asthma remains unknown.Interestingly, while most individuals experience similar environmentalexposures, only certain individuals develop atopic allergy and asthma.This hypersensitivity to environmental allergens known as “atopy” isoften indicated by elevated serum IgE levels or abnormally great skintest response to allergens in atopic individuals as compared tononatopics.¹⁰ Strong evidence for a close relationship between atopicallergy and asthma is derived from the fact that most asthmatics haveclinical and serologic evidence of atopy.⁴⁻⁹ In particular, youngerasthmatics have a high incidence of atopy.¹⁰ In addition, immunologicfactors associated with an increase in serum total IgE levels are veryclosely related to impaired pulmonary function.³

Both the diagnosis and treatment of these disorders are problematic.¹The assessment of inflamed lung tissue is often difficult, andfrequently the source of the inflammation cannot be determined. Withoutknowledge of the source of the airway inflammation and protection fromthe inciting foreign environmental agent or agents, the inflammatoryprocess cannot be interrupted. It is now generally accepted that failureto control the pulmonary inflammation leads to significant loss of lungfunction over time.

Current treatments suffer their own set of disadvantages. The maintherapeutic agents, β agonists, reduce the symptoms, i.e., transientlyimprove pulmonary functions, but do not affect the underlyinginflammation so that lung tissue remains in jeopardy. In addition,constant use of β agonists results in desensitization which reducestheir efficacy and safety.² The agents that can diminish the underlyinginflammation, the anti-inflammatory steroids, have their own known listof disadvantages that range from immunosuppression to bone loss.²

Because of the problems associated with conventional therapies,alternative treatment strategies have been evaluated.⁶⁵⁻⁶⁶ GlycophorinA,⁶⁴ cyclosporin,⁶⁵ and a nonapeptide fragment of IL-2,⁶³ all inhibitinterleukin-2 dependent T lymphocyte proliferation and therefore, IL-9production,⁵¹ however, they are known to have many other effects.² Forexample, cyclosporin is used as a immunosuppressant after organtransplantation. While these agents may represent alternatives tosteroids in the treatment of asthmatics,⁶³⁻⁶⁶ they inhibit interleukin-2dependent T lymphocyte proliferation and potentially critical immunefunctions associated with homeostasis. What is needed in the art is theidentification of a pathway critical to the development of asthma thatexplains the episodic nature of the disorder and the close associationwith allergy that is downstream of these critical immune functions.Nature demonstrated that this pathway is the appropriate target fortherapy since biologic variability normally exists at this pathway andthese individuals are otherwise generally not immunocompromised or illexcept for their symptoms of atopy.

Because of the difficulties related to the diagnosis and treatment ofasthma, the complex pathophysiology of this disorder is under intensivestudy. Although this disorder is heterogeneous and may be difficult todefine because it can take many forms, certain features are found incommon among asthmatics. Examples of such traits include elevated serumIgE levels, abnormal skin test response to allergen challenge, bronchialhyperresponsiveness [BHR], bronchodilator reversibility, and airflowobstruction.³⁻¹⁰ These expressions of these asthma related phenotypesmay be studied as quantitative or qualitative measures.

Elevated IgE levels are also closely correlated with BHR, a heightenedbronchoconstrictor response to a variety of stimuli.^(4,6,8,9) BHR isbelieved to reflect the presence of airway inflammation,^(6,8) and isconsidered a risk factor for asthma.¹¹⁻¹² BHR is accompanied bybronchial inflammation and an allergic diathesis in asthmaticindividuals.¹³⁻²¹ Even in children with no symptoms of atopy and asthma,BHR is strongly associated with elevated IgE levels.¹⁹

A number of studies document a heritable component to atopy andasthma.^(4,10,21) However, family studies have been difficult tointerpret since these disorders are significantly influenced by age andgender, as well as many environmental factors such as allergens, viralinfections, and pollutants.²²⁻²⁴ Moreover, because there is no knownbiochemical defect associated with susceptibility to these disorders,the mutant genes and their abnormal gene products can only be recognizedby the anomalous phenotypes they produce. Thus, an important first stepin isolating and characterizing a heritable component is identifying thechromosomal locations of the genes.

Cookson et al. provided the first description of a genetic localizationfor inherited atopy.²⁵ These investigators described evidence forgenetic linkage between atopy and a single marker on a specificchromosomal region designated 11q13.1. Later, they suggested evidence ofmaternal inheritance for atopy at this locus.²⁶ Although maternalinheritance [genetic imprinting] had been observed for atopy, it hadnever been explained previously. However, efforts to confirm thislinkage have not been generally successful.²⁷⁻³¹

Recently, the β subunit of the high-affinity IgE receptor was mapped tochromosome 11q, and a putative mutation associated with atopy has beendescribed in this gene.^(32,33) However, because of the difficulties byothers of replicating this linkage, the significance of this gene andpolymorphism remains unclear. While additional studies will be requiredto confirm whether this putative mutation causes atopy in the generalpopulation, data collected so far suggests this polymorphism is unlikelyto represent a frequent cause of atopy.

Because serum IgE levels are so closely associated with the onset andseverity of allergy and asthma as clinical disorders, attention hasfocused on studies of the genetic regulation of serum total IgE levels.While past studies have provided evidence for Mendelian inheritance forserum total IgE levels,³⁴⁻³⁸ an indication of the existence of oneregulatory gene, others have found evidence for polygenic inheritance ofIgE, i.e., existence of several responsible genes.³⁹

Artisans have found several genes that may be important in theregulation of IgE and the development or progression of bronchialinflammation associated with asthma on chromosome 5q. They include genesencoding several interleukins, such as IL-3, IL-4, IL-5, IL-9, IL-13,granulocyte macrophage colony stimulating factor [GM CSF], a receptorfor macrophage colony stimulating factor [CSF-1R], fibroblast growthfactor acidic [FGFA], as well as others.⁴⁰ Recent evidence from familystudies suggests genetic linkage between serum IgE levels and DNAmarkers in the region of these candidate genes on chromosome 5q.^(41,42)Together, these investigations suggest that one or more major genes inthe vicinity of the interleukin complex on chromosome 5q regulates asignificant amount of the observed biologic variability in serum IgEthat is likely to be important in the development of atopy and asthma.

Linkage [sib-pair analyses] was also used previously to identify agenetic localization for BHR.⁷⁹ Because BHR was known to be associatedwith a major gene for atopy, chromosomal regions reported to beimportant in the regulation of serum IgE levels were examined.⁴²Candidate regions for atopy have been identified by linkage analyses.These studies identified the existence of a major gene for atopy onhuman chromosome 5q31-q33.⁴²

Therefore, to determine the chromosomal location of a gene[s] providingsusceptibility to BHR, which would be coinherited with a major gene foratopy, experiments were carried out using linkage analyses between BHRand genetic markers on chromosome 5q.^(42,79,82) Individuals with BHRwere identified by responsiveness to histamine. Markers useful formapping asthma-related genes are shown in FIG. 1.

Specifically, gene candidates for asthma, bronchial hyperresponsiveness,and atopy are shown [right] in their approximate location relative tothe markers shown. The map includes the interleukin genes IL-4, IL-13,IL-5, and IL-3; CDC25, cell division cycle-25; CSF2,granulocyte-macrophage colony stimulating factor [GMCSF]; EGR1 earlygrowth response gene-1; CD14, cell antigen 14; ADRB2, the β2-adrenergicreceptor; GRL1, lymphocyte-specific glucocorticoid receptor; PDGFR,platelet-derived growth factor receptor. Bands 5q31-q33 extendapproximately from IL-4 to D5S410. The distances reported aresex-averaged recombination fractions.

Affected sib-pair analyses demonstrated statistically significantevidence for linkage between BHR and D5S436, D5S658, and several othermarkers located nearby on chromosome 5q31-q33.⁷⁹ These data stronglysupported the hypothesis that one or more closely spaced gene[s] onchromosome 5q31-q33 determine susceptibility to BHR, atopy, andasthma.^(79,80,81,82)

Recently linkage has also been demonstrated between the asthma phenotypeand genetic markers on chromosome 5q31-q33.⁸³ This region of the humangenome was evaluated for linkage with asthma because of the large numberof genes representing reasonable positional candidates for providinggenetic susceptibility for atopy and BHR.

Linkage was demonstrated using the methods described above.^(42,83)Specifically, 84 families were analyzed from the Netherlands with bothsib-pair and LODs for markers from this same region of chromosome 5qpreviously shown to be linked to BHR and atopy.^(42,83) An algorithm wasused to categorize obstructive airways disease in the asthmatic probandsand their families. This classification scheme was based, as describedpreviously, on the presence or absence of BHR to histamine, respiratorysymptoms, significant smoking history [>5 pack years), atopy as definedby skin test response, airway obstruction (FEV1 % predicted<95% CI] andreversibility to a bronchodilator [>9% predicted].

Evidence was found for linkage between asthma and markers on chromosome5q by affected sib pair analysis (N=10, P<0.05) and by maximumlikelihood analysis with a dominant model for the asthma phenotype.⁸³

Asthma was linked to D5S658 with a maximal LOD of 3.64 at θ=0.03, usinga dominant model [class 1 affected, class 2-4 uncertain, class 5unaffected] with a gene frequency of 0.015 [prevalence of 3%]. A maximalLOD of 2.71 at θ=0.0 was observed for D5S470 which is approximately 5 cMtelomeric, or away from IL-9, relative to D5S436.⁸³

Subsequent to the original filing of this application, IL-9 or a genenearby was suggested as likely to be important use atopy and asthma.⁴³The IL-9 suggestion was based on a strong correlation in a randomlyascertained population between log serum total IgE levels and alleles ofa genetic marker in the IL-9 gene.⁴³ This type of association with oneor more specific alleles of a marker is termed “linkage disequilibrium”,and generally suggests that a nearby gene determines the biologicvariability under study.⁴⁴

The IL-9 gene has been mapped to the q31-q33 region of chromosome 5.⁴⁰Only a single copy of the gene is found in the human genome.^(45,46)Structural similarity has been observed for the human and murine IL-9genes.^(45,46) Each gene consists of five exons and four intronsextending across approximately four Kb of DNA. Expression of the geneappears to be restricted to activated T cells.^(45,46)

The functions of IL-9 now extend well beyond those originallyrecognized. While IL-9 serves as a T cell growth factor, this cytokineis also known to mediate the growth of erythroid progenitors, B cells,mast cells, and fetal thymocytes.^(45,46) IL-9 acts synergistically withIL-3 in causing mast cell activation and proliferation.⁴⁷ This cytokinealso potentiates the IL-4 induced production of IgE, IgG, and IgM bynormal human B lymphocytes.⁴⁸ IL-9 also potentiates the IL-4 inducedrelease of IgE and IgG1 by murine B lymphocytes.⁴⁹ A critical role forIL-9 in the mucosal inflammatory response to parasitic infection hasalso been demonstrated.^(50,51)

In addition to IL-9, chromosome 5q bears numerous other gene candidatesincluding IL-3, IRF1, EGR1, ITK, GRL1, ADRB2, CSF1R, FGFA, ITGA2, CD14,PDGFR, CDC25, CSF2, IL-4, IL-5, IL-12B, and IL-13. These may all beimportant in atopic allergy and as potential targets for therapeuticdevelopment. Moreover, the art lacks any knowledge regarding how thesequence of IL-9 or the function of IL-9 specifically correlates withatopic allergy, asthma, or bronchial hyperresponsiveness. Without suchknowledge, artisans would not know how or whether to use IL-9 to eitherdiagnose or treat these disorders.

The art does provide that IL-9 is a novel cytokine having an apparentmolecular weight of approximately between 20 to 30 kD as determined bysodium dodecyl sulfate polyacrylamide gel electrophoresis under reducingconditions. It is produced as a 144 amino acid protein, that isprocessed to a 126 amino acid glycoprotein. Yang et al.⁸⁵ disclose thatthe DNA sequence encoding IL-9 comprises approximately 630 nucleotides,with approximately 450 nucleotides in the proper reading frame for theprotein.

It is also known in the art that multiple protein isoforms may begenerated from a single genetic locus by alternative splicing.Alternative splicing is an efficient mechanism by which multiple proteinisoforms may be generated from a single genetic locus. Alternativesplicing is used in terminally differentiated cells to reversibly modifyprotein expression without changing the genetic content of the cells.These protein isoforms are preferentially expressed in different tissuesor during different states of cell differentiation or activation.Protein isoforms may have different functions and Alms and White havecloned and expressed a naturally occurring splice variant of IL-4,formed by the omission of exon 2, thus called IL-4δ2.⁸⁶ It was observedthat IL-4δ2 inhibits T-cell proliferation induced by IL-4.

However, the art lacks any knowledge about IL-9 protein isoforms whichare formed by deletions of exons 2 and 3 or the regulatory functionsexhibited by these truncated proteins. Specifically, their role inregulating the biological activity, namely, the down-regulation of IL-9expression or activity is unclear. Moreover, the formation of suchisoforms by alternative splicing has not been previously observed orused to provide variants of IL-9 which function as agonists orantagonists of the native cytokine.

The art also lacks any knowledge about the role of the IL-9 receptorwith asthma-related disorders. It is known that IL-9 binds to a specificreceptor expressed on the surface of target cells.^(46,52,53) Thereceptor actually consists of two protein chains: one protein chain,known as the IL-9 receptor, binds specifically with IL-9 and the otherprotein chain is the chain, which is shared in common with the IL-2receptor.⁴⁶ In addition, the human IL-9 receptor cDNA has beencloned.^(46,52,53) This cDNA encodes a 522 amino acid protein whichexhibits significant homology to the murine IL-9 receptor. Theextracellular region of the receptor is highly conserved, with 67%homology existing between the murine and human proteins. The cytoplasmicregion of the receptor is less highly conserved. The human cytoplasmicdomain is much larger than the corresponding region of the murinereceptor.⁴⁶

The IL-9 receptor gene has also been characterized.⁵³ It is thought toexist as a single copy in the mouse genome and is composed of nine exonsand eight introns.⁵³ The human genome contains at least four IL-9receptor pseudogenes. The human IL-9 receptor gene has been mapped tothe 320 kb subtelomeric region of the sex chromosomes X and Y.⁴⁶Nonetheless, despite these studies, the art lacks any knowledge of arelation between the IL-9 receptor and atopic allergy, asthma, orbronchial hyperresponsiveness.

Thus, the art lacks any knowledge of how the IL-9 gene, its receptor,and their functions, are related to atopic allergy, asthma, bronchialhyperresponsiveness, and related disorders. Therefore, there is aspecific need in the art for genetic information on atopic allergy,asthma, bronchial hyperresponsiveness, and for elucidation of the roleof IL-9 in the etiology of these disorders. There is also a need forelucidation of the role of the IL-9 receptor and the IL-9 receptor genein these disorders. Furthermore, most significantly, based on thisknowledge, there is a need for the identification of agents which arecapable of regulating the interaction between IL-9 and its receptor fortreating these disorders.

SUMMARY OF THE INVENTION

Applicant has satisfied the long felt need for a treatment for atopicallergy including asthma and related disorders by providing informationdemonstrating the role of IL-9 (also known as Asthma Associated Factor1, or AAFI) in the pathogenesis of these disorders which information hasled to compounds that are capable of regulating the activity of IL-9.Applicant has also demonstrated conserved linkage and synteny homologiesbetween mice and humans for a gene that determines biologic variabilityin airway hyperresponsiveness. These relationships specifically identifyIL-9 as a gene candidate. In addition, applicant has determined thatIL-9 is critical to a number of antigen-induced responses in miceincluding bronchial hyperresponsiveness, eosinophilia and elevated cellcounts in bronchial lavage, and elevated serum total IgE. These findingstypify the allergic inflammation associated with asthma.

Furthermore, applicant has determined that a C to T nucleic acidvariation at position 3365 in exon 5 of the human IL-9 gene produces thepredicted amino acid substitution of a methionine for a threonine atcodon 117 of IL-9. When this substitution occurs in both alleles in oneindividual, it is associated with less evidence of atopic allergyincluding asthma, fewer abnormal skin test responses, and a lower serumtotal IgE. Thus, applicant has identified the existence of anonasthmatic, nonatopic phenotype characterized by methionine at codon117 when it occurs in both IL-9 gene products in one individual. As anadditional significant corollary, applicant has identified the existenceof susceptibility to an asthmatic, atopic phenotype characterized by athreonine at codon 117. Thus, the invention includes purified andisolated DNA molecules having such a sequence as well as the peptidesencoded by such DNA.

The biological activity of IL-9 results from its binding to the IL-9receptor and the consequent propagation of a regulatory signal inspecific cells. Therefore, IL-9 functions can be interrupted orregulated by the interaction of IL-9 agonists or antagonists with IL-9or its receptor. Down regulation, i.e. reduction of the functionscontrolled by IL-9, is achieved in a number of ways. Administeringagonists or antagonists that can interrupt the binding of IL-9 to itsreceptor is one key mechanism and such agonists and antagonists arewithin the claimed invention. Examples include administration ofpolypeptide products encoded by the DNA sequences of IL-9 or IL-9receptor wherein the DNA sequences contain various mutations. Thesemutations may be point mutations, insertions, deletions, or splicedvariants of IL-9 or its receptor.

A further embodiment of this invention includes the regulation of theactivity of IL-9 by administering “agonists and antagonists.” Theskilled artisan will readily recognize that all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention. Specifically, residues 43-60 and 71-90 of the matureprotein appear to be important for receptor binding. Applicant has shownthat peptides KP-16 (residues 43-60) and KP-20 (residues 71-90) act asreceptor antagonists. In addition, these residues in the native IL-9molecule are predicted to form anti-parallel helical structures. Thethree dimensional structure of the protein suggests that specificallyserine 52 and/or glutamic acid 53 interact with lysine 85, serine 56interacts with lysine 82, and threonine 59 interacts with valine 78. Thethree dimensional coordinates of these anti-parallel helices and therelated functional groups represent the requisite 3-dimensionalconformation critical for receptor binding and compounds which simulatethese relationships are within the scope of this invention.

The biological activity of the IL-9 receptor (also called AsthmaAssociates Factor 2, AAF2) can also be modulated by using soluble IL-9receptor molecules. Such a molecule prevents the binding of IL-9 to thecell-bound receptor and acts as an antagonist for IL-9, and is alsowithin the scope of this invention.

Polyclonal and monoclonal antibodies which block the binding of IL-9 toits receptor are also within the scope of this invention and are usefultherapeutic agents in treating atopic allergy including asthma andrelated disorders.

Another embodiment of this invention relates to the use of isolated DNAsequences containing various mutations such as point mutations,insertions, deletions, or spliced mutations of IL-9 or the IL-9 receptorin gene therapy.

Expression of IL-9 and IL-9 receptor is also down-regulated byadministering an effective amount of synthetic antisense oligonucleotidesequences. The oligonucleotide compounds of the invention bind to themRNA coding for human IL-9 and IL-9 receptor thereby inhibitingexpression of these molecules.

The structure of both IL-9 and the IL-9 receptor have been examined andanalyzed in great detail and amino acid residues of IL-9 critical forreceptor binding have been identified. Based on structural studies andthe binding characteristics of this specific binding pair, thisinvention further includes small molecules tailored such that theirstructural conformation provides the residues essential for blocking theinteraction of IL-9 with the IL-9 receptor. Such blockade results inmodulation of the activity of the receptor and these molecules are,therefore, useful in treating atopic allergies.

Another embodiment of this invention is directed to the regulation ofdownstream signaling pathways necessary for IL-9 function. IL-9 inducestyrosine phosphorylation of Stat3 which appears to be unique to the IL-9signaling pathway⁵⁸ and is useful as a target for inhibitors. Specificand nonspecific inhibitors of tyrosine kinase such as tyrophostins are,therefore, useful in downstream regulation of the physiological activityof IL-9, and are part of the invention.

In a further embodiment aminosterol compounds are also useful intreating atopic allergies and related disorders because they are alsoinvolved in blocking signal transduction of the IL-9 signal transductionpathway.

The products discussed above represent various effective therapeuticagents in treating atopic allergies, asthma and other related disorders.

This invention also includes the truncated polypeptides encoded by theDNA molecules described above. These polypeptides are capable ofregulating the interaction of IL-9 with the IL-9 receptor.

Thus, applicant has identified the critical role of the IL-9 pathway inpathogenesis of atopic allergy, including bronchial hyperresponsiveness,asthma, and related disorders. More specifically, applicant has providedantagonists and methods of identifying antagonists that are capable ofregulating the interaction between IL-9 and its receptor. Applicant alsoprovides methods for regulating the activity of IL-9 by: 1)administering a compound having activity comparable to IL-9 containingmethionine at codon 117 and the ability to bind to a receptor for IL-9in an amount sufficient to down-regulate the activity of IL-9; and 2) byadministering truncated protein products encoded by isolated nucleicacid sequences comprising deletions of any one or more of exons 1, 2, 3,4, or 5.

Having identified the critical role of the IL-9 pathway in atopicallergy, bronchial hyperresponsiveness, and asthma, applicant alsoprovides a method for the diagnosis of susceptibility to atopic allergy,asthma, and related disorders. Lastly, applicant provides a method forassaying the functions of IL-9 and its receptor to identify compounds oragents that may be administered in an amount sufficient to down-regulateeither the expression or functions of IL-9 and the IL-9 receptor.

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and, together with the description, serve to explain theprinciple of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map showing the relative order and distance in centiMorgans [cM]between the polymorphic genetic markers useful for mappingasthma-related genes.

FIG. 2: Illustration of the genetic map of human chromosome 5q31-q33 andsyntenic regions in the mouse.

FIG. 3: The LOD score curve on mouse chromosome 13 foratracurium-induced airway responsiveness in mice with increasedsusceptibility to bronchoconstrictor stimuli.

FIG. 4: Alignment of amino acid sequences corresponding to exon 5 of thehuman and murine IL-9 genes. The first sequence is translated from theThr allele of the human gene. The middle sequence is translated from theMet allele of the human gene. The final sequence is translated from themurine gene.

FIG. 5: Histogram of the correlation between human IL-9 gene alleles andserum total IgE titers measured in international units. S/S denotesThr/Thr individuals, S/R denotes Thr/Met individuals and R/R denotesMet/Met individuals.

FIG. 6: Illustration the simple sequence repeat polymorphism at the IL-9locus.

FIG. 7: Translated cDNA sequence of Thr117 version of IL-9.

FIG. 8: Translated cDNA sequence of Met117 version of IL-9.

FIG. 9: Map of pFlag expression construct with Thr117.

FIG. 10: Sequence of pFlag expression construct for the Thr117 versionof the cDNA from the region surrounding the site of ligation.

FIG. 11: Map of pFlag expression construct with Met117.

FIG. 12: Sequence of pFlag expression construct for the Met117 versionof the cDNA from the region surrounding the site of ligation.

FIG. 13: Western blot of recombinant IL-9 proteins

FIG. 14: Amino acid sequences for inhibitory peptides.

FIG. 15: Inhibition by KP-16 of IL-9 mediated MO7e proliferation.

FIG. 16: Inhibition by KP-20 of IL-9 mediated MO7e proliferation.

FIG. 17: Inhibition by KP-23 of IL-9 mediated MO7e proliferation.

FIG. 18: Inhibition by various tyrophostins of IL-9 mediated MO7eproliferation.

FIG. 19: Inhibition by various aminosterols of IL-9 mediated MO7eproliferation.

FIG. 20: Characterization of the role of IL-9 in the antigen response invivo.

FIG. 21: Histologic examination of lungs from control, ova challenged,and anti-IL-9 pretreated animals.

FIG. 22: Inhibition of the antigen response in vivo by blockingantibodies to the murine IL-9 receptor.

FIG. 24: Expression of human Met117 IL-9 and Thr117 IL-9.

FIG. 25: Binding of the human recombinant Met117 and Thr117 forms ofIL-9 to a soluble receptor.

FIG. 26: Steady state levels of IL-9 in unstimulated and stimulatedmurine splenocytes.

FIG. 27: An appendix of chemical moieties.

FIG. 28: Aminosterols isolated from the dog fish shark.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has resolved the needs in the art by elucidating an IL-9pathway and compositions that affect that pathway that may be used inthe diagnosis, prevention or treatment of atopic allergy includingasthma and related disorders. Asthma encompasses inflammatory disordersof the airways with reversible airflow obstruction. Atopic allergyrefers to atopy, and related disorders including asthma, bronchialhyperresponsiveness (BHR), rhinitis, urticaria, allergic inflammatorydisorders of the bowel, and various forms of eczema. Atopy is ahypersensitivity to environmental allergens expressed as the elevationof serum total IgE or abnormal skin test responses to allergens ascompared to controls. BHR refers to bronchial hyperresponsiveness, aheightened bronchoconstrictor response to a variety of stimuli.

By analyzing the DNA of families that exhibit asthma-related disorders,applicant has identified a polymorphism in the IL-9 gene that correlateswith the biologic variability of serum total IgE as one measurableexpression of atopy. The IL-9 gene (also known as Asthma AssociatedFactor 1 or AAF1) refers to the genetic locus of interleukin-9, acytokine exhibiting a variety of functions involving the regulation ofhuman myeloid and lymphoid systems. The IL-9 gene of the presentinvention is found in the q31-q33 region of human chromosome 5 andchromosome 13 in the mouse.

By polymorphism, applicant means a change in a specific DNA sequence,termed a “locus”, from the prevailing sequence. In general, a locus isdefined as polymorphic when artisans have identified two or more allelesencompassing that locus and the least common allele exists at afrequency of 1% or more.

The polymorphism of the present invention leads to an amino acidsubstitution at residue 117 of IL-9. Specifically, instead of thehydrophilic amino acid threonine, the IL-9 of the present inventionexhibits the hydrophobic amino acid methionine (Met IL-9). On a geneticlevel, the polymorphism of the present invention is a substitution of athymine residue for a cytosine residue at nucleotide position 3365 inthe human IL-9 gene as it is described by Renauld and colleagues (1990)[GenBank accession numbers M30135 and M30136],⁵⁴ or at the comparablenucleotide position 4244 of the human IL-9 gene sequence reported byKelleher et al.,(1991) [GenBank accession number M86593].⁵⁵

Individuals with a threonine (Thr) at amino acid 117 of IL-9 in eitherone or both of their alleles (Thr/Thr or Thr/Met) generally exhibitsusceptibility to an asthmatic or atopic allergic phenotype, and thesegenotypes are characterized by higher mean serum total IgE levels in thepopulations studied. In contrast, those individuals with a methionine(Met) at codon 117 of IL-9 in both alleles (Met/Met) exhibit a lack ofasthma, fewer abnormal skin test responses, and a lower serum total IgE.Thus, the Met/Met genotype of IL-9 appears to protect against asthma oratopic allergy.

Accordingly, the invention provides a purified and isolated DNA moleculecomprising a nucleotide sequence encoding human interleukin 9 containingmethionine at position 117 (Met IL-9), or a fragment thereof. Theinvention also includes degenerate sequences of the DNA as well assequences that are substantially homologous. The source of the IL-9 ofthe invention is human. Alternatively, the DNA or fragment thereof maybe synthesized using methods known in the art. It is also possible toproduce the compound by genetic engineering techniques, by constructingDNA by any accepted technique, cloning the DNA in an expression vehicleand transfecting the vehicle into a cell which will express thecompound. See, for example, the methods set forth in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. Cold Spring HarborLaboratory Press [1985].

Airway hyperresponsiveness is found in virtually all asthmatics and insome strains of inbred mice (DBA/2).⁸⁴ Airway hyperresponsiveness is arisk factor for the development of asthma in humans and is used inanimal models of asthma as a physiologic measure to assess the efficacyof treatment for asthma. These data along with human⁷⁹ and murinegenetic mapping results (see Examples 1 and 2) suggest a critical rolefor the murine IL-9 gene product in the airway response of the mouse. Inparticular, the hyperresponsive DBA/2(D2) mice differ from theC57BL/6(B6) hyporesponsive mice⁸⁴ in their expression of steady statelevels of IL-9 (See Example 14, FIG. 26). Furthermore, pretreatment withblocking antibodies to IL-9/IL-9 receptor can optionally providecomplete protection from antigen induced airway hyperresponsiveness andinflammation in mice demonstrating a critical regulatory role for IL-9in these immune responses. Thus, these data demonstrate that althoughdifferent molecular changes produce biologic variability in airwayresponsiveness in humans and mice, these changes arise in the samegene(s) (IL-9/IL-9R) that regulate this pathway. Taken together, theseobservations confirm the critical role of IL-9 and the IL-9 receptor inairway hyperresponsiveness, asthma, and atopic allergy. Moreover, thesedata demonstrate that agents of the convention, which block theinteraction of IL-9 with its receptor, protect against an antigeninduced response such as those detailed above.

Further evidence defining the critical role of IL-9 in the pathogenesisof atopic allergy, bronchial hyperresponsivenss, asthma, and relateddisorders derives directly from the applicants observation that IL-9 iscritical to a number of antigen induced responses in mice. When thefunctions of IL-9 are down regulated by antibody pretreatment prior toaerosol challenge with antigen, the animals can be completely protectedfrom the antigen induced responses. These responses include: bronchialhyperresponsiveness, eosinophilia and elevated cell counts in bronchiallavage, histologic changes in lung associated with inflammation, andelevated serum total IgE. Thus, the treatment of such responses, whichare critical to the pathogenesis of atopic allergy and whichcharacterize the allergic inflammation associated with asthma, by thedown regulation of the functions of IL-9, are within the scope of thisinvention.

Applicant also teaches the regulation of the activity of IL-9 byadministering “agonists and antagonists” to the IL-9 receptor. Theskilled artisan will readily recognize that all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention. Applicant has shown that peptides KP-16 (IL-9residues 43-60) and KP-20 (IL-9 residues 71-90) (produced using standardpeptide automated synthesis techniques, for example, the AppliedBiosystems Model 431A Peptide Synthesizer) act as IL-9 antagonists.Specifically, applicant demonstrates that residues 43-60 and 71-90 ofthe mature protein appear to be important for receptor binding. Inaddition, these residues include most of exon 4 (amino acids 44-88) andare predicted to form anti-parallel helical structures. The threedimensional structure of the protein suggests that specifically serine52 and/or glutamic acid 53 interact with lysine 85, serine 56 interactswith lysine 82, and threonine 59 interacts with valine 78. The threedimensional coordinates of these parallel helices and the relatedfunctional groups represent the requisite 3-dimensional conformationcritical for receptor binding and compounds that simulate theserelationships are within the scope of this invention.

The demonstration of an IL-9 sequence associated with an asthma-likephenotype, and one associated with the lack of an asthma-like phenotype,indicates that the lungs' inflammatory response to antigen is dependenton IL-9, and therefore, that down regulating the function of IL-9 shouldprotect against the antigen induced response. Furthermore, applicantalso provides methods of diagnosing susceptibility to atopic allergy andrelated disorders and for treating these disorders based on therelationship between IL-9 and its receptor.

A receptor is a soluble or membrane bound component that recognizes andbinds to molecules, and the IL-9 receptor (also known as AsthmaAssociated Factor 2 or AAF2) of the invention is the component thatrecognizes and binds to IL-9. The functions of the IL-9 receptor consistof binding an IL-9-like molecule and propagating its regulatory signalin specific cells.⁵⁷⁻⁶⁰ An interruption of that function will lead to adown regulation, i.e., reduction, of either the expression of IL-9 or ofthe functions controlled by IL-9. Accordingly, by virtue of thisinteraction between. IL-9 and the IL-9 receptor, certain functions ofthe organism are modulated or controlled. For a general discussion ofreceptors, see Goodman and Gilman's The Pharmacologic Basis ofTherapeutics (Seventh Edition, MacMillan Publishing Company, N.Y. USA,1985).

One diagnostic embodiment involves the recognition of variations in theDNA sequence of IL-9. One method involves the introduction of a nucleicacid molecule (also known as a probe) having a sequence complementary tothe IL-9 of the invention under sufficient hybridizing conditions, aswould be understood by those in the art. In one embodiment, the sequencewill bind specifically to the Met117 IL-9 or to Thr117 IL-9, and inanother embodiment will bind to both Met117 IL-9 and Thr117 IL-9.Another method of recognizing DNA sequence variation associated withthese disorders is direct DNA sequence analysis by multiple methods wellknown in the art.⁷⁷ Another embodiment involves the detection of DNAsequence variation in the IL-9 gene associated with thesedisorders.⁷³⁻⁷⁷ These include the polymerase chain reaction, restrictionfragment length polymorphism (RFLP) analysis and single strandedconformational analysis. In a preferred embodiment, applicant providesspecifically for a method to recognize, on a genetic level, thepolymorphism in IL-9 associated with the Thr and Met alleles using aStyI RFLP as described herein. In other embodiments Nla, Pfim1, PflM1,and Nco1 RFLPs may be used to distinguish these two alleles of IL-9genes.

Another embodiment involves treatment of atopic allergy and relateddisorders. In a preferred embodiment, the applicant provides a method ofadministering a compound having activity comparable to Met IL-9 and theability to bind to an IL-9 receptor in an amount sufficient to downregulate the activity of IL-9. A compound having activity comparable toMet IL-9 is a compound that functions similarly but not necessarilyidentically. Thus, it may bind to the IL-9 receptor but without the samephysiological effects. Examples include amino acid sequences of IL-9containing various point mutations and/or deletions and sequencessubstantially homologous thereto. For example, such a compound mayinterrupt the binding of Thr IL-9 to the IL-9 receptor as measured bytechniques known in the art. The invention also encompasses functionallyeffective fragments of the above amino acid sequences. In one suchtechnique, the Thr IL-9 may be considered a “ligand” for the IL-9receptor, and binding between the two may be assessed by ligand-bindingassays which are well known in the art as set forth in Goodman andGilman's The Pharmacologic Basis of Therapeutics (Seventh Edition,MacMillan Publishing Company, N.Y. USA, 1985).

In another embodiment, the compound may resemble the Met allele of IL-9in structure. Thus, such a compound may incorporate a methionine incodon 117 of IL-9 or may incorporate another hydrophobic amino acid.Thus, included within the scope of this invention are IL-9 varientscomprising substitutions of Thr at position 117 by amino acids selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophan, and methionine. Alternatively, thecompound of the invention may exist as a fragment of IL-9 with astructural composition similar to Met IL-9. In another embodiment of theinvention, the compound may retain functions comparable to Met IL-9, butmay not resemble Met IL-9 in structure. For example, the composition ofthe compound may include molecules other than amino acids. This exampleis merely illustrative and one of ordinary skill in the art wouldreadily recognize that other substitutions and/or deletion analogs ofIL-9 resulting in effective antagonists are also within the scope ofthis invention. As discussed above all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention.

Specific assays may be based on IL-9's known regulation, in part, of theproliferation of T lymphocytes, IgE synthesis, and release from mastcells.⁵⁴⁻⁶⁰ Another assay involves the ability of human IL-9 tospecifically induce the rapid and transient tyrosine phosphorylation ofmultiple proteins in M07e cells.⁵⁷ Because this response is dependent onthe expression and activation of the IL-9 receptor, it represents asimple method or assay for the characterization of potentially valuablecompounds. The tyrosine phosphorylation of Stat3 transcriptional factorappears to be specifically related to the actions of IL-9,⁵⁸ and thisresponse represents a simple method or assay for the characterization ofcompounds within the invention. Still another method to characterize thefunction of IL-9 and IL-9-like molecules involves the well known murineTS1 clone and the D10 clone available from ATCC used to assess humanIL-9 function with a cellular proliferation assay.⁵⁹ The Met IL-9 thatforms a part of the invention may be viewed as a “weak agonist” of theIL-9 receptor. Such weak agonists are another preferred embodiment ofthe invention. The term agonist, according to this invention, includescompounds that mimic at least some of the effects of endogenouscompounds by interacting or binding with a receptor. Agonists thatinteract or bind to the IL-9 receptor on the surface of certain cellsinitiate a series of biochemical and physiological changes that arecharacteristic of this cytokine's actions.^(45-51,54-60) To identifyother weak agonists of the invention, one may test for binding to theIL-9 receptor or for IL-9-like functions as described herein and in thecited literature^(2,45-51,54-60)

The present invention also includes antagonists of IL-9 and itsreceptor. Antagonists are compounds that are themselves devoid ofpharmacological activity but cause effects by preventing the action ofan agonist. To identify an antagonist of the invention, one may test forcompetitive binding with a known agonist or for down-regulation ofIL-9-like functions as described herein and in the citedliterature.^(2,45-51,54-60)

The binding of either the agonist or antagonist may involve all knowntypes of interactions including ionic forces, hydrogen bonding,hydrophobic interactions, van der Waals forces, and covalent bonds. Inmany cases, bonds of multiple types are important in the interaction ofan agonist or antagonist with a receptor.

In a further embodiment, these compounds may be analogs of IL-9. IL-9analogs may be produced by point mutations in the isolated DNA sequencefor the gene, nucleotide substitutions, and/or deletions which can becreated by methods that are all well described in the art.⁶² Thisinvention also includes spliced variants of IL-9 and discloses isolatednucleic acid sequences of interleukin-9, which contain deletions of oneor more of its five exons. The term “spliced variants” as used hereindenotes a purified and isolated DNA molecule encoding human IL-9comprising at least one exon. There is no evidence of naturallyexpressed spliced mutants in the art. Thus, the present inventionprovides an isolated nucleic acid containing exons 1, 4 and 5 of humanIL-9. Other variants within the scope of this invention includesequences comprising exons 1, 2, 3, 4, and 5; exons 1, 2, 3, and 4;exons 1, 2, 4, and 5 and exons 1, 3, 4, and 5. It must be understoodthat these exons may contain various point mutations.

Specific examples of antagonistic peptides derived from IL-9 includeKP-16 (SEQ. ID No. 13) and KP-20 (SEQ. ID NO. 14) which are derived fromexon 4. Exon 4 encodes 44 amino acids while the peptides mentioned abovecontain 16 and 20 amino acids respectively and they do not overlap.These peptides exhibit considerable inhibitory activity bothindividually and when assayed in combination. Additionally, KP-23 (SEQID NO. 15) and KP-24 (SEQ ID NO 16) are derived from exon 5 and alsoexhibit similar activity. Splice variants of IL-9 can be formed bydeletion of any one or more of the IL-9 exons 1 through 5. As shownabove, peptides derived from these exons show regulatory capability and,accordingly, are useful in treating atopic allergies, including asthma.

It is known in the art that, in multienzyme systems, the first orregulatory enzyme can be activated or inhibited by the end product ofthe multi-enzyme system. When the concentration of the end productincreases over the steady state concentration, the end product will actas a specific activator or inhibitor of the regulatory enzyme in thesequence. Such feedback mechanism is also relevant to the IL-9 systemand it is observed that the various polypeptides of this invention arecapable of exerting such activation or inhibitory control on theactivity of the IL-9 receptor and possibly the expression or function ofother cytokines and their receptors that play a role in the pathogenesisof asthma.

The invention also includes modifications of agonists or antagoniststhat can be made using knowledge that is routine to those in this art.For example, the affinity of a compound for a receptor is generallyclosely related to the chemical structure of the compound. Thus,structure-activity relationships may be used to modify the agonists andantagonists of the invention. For example, the techniques ofcrystallography/X-ray diffraction and NMR may be used to makemodifications of the invention.

For example, one can create a three dimensional structure of human IL-9that can be used as a template for building structural models ofdeletion mutants using molecular graphics. These models can then be usedto identify and construct a mutant IL-9 molecule with affinity for theIL-9 receptor comparable to IL-9, but with a lower biologic activity.What is meant by lower biologic activity is 2 to 100,000 fold less thanIL-9, preferably 100 to 1,000 fold less than IL-9.

In still another embodiment, these compounds also may be used as dynamicprobes for receptor structure and to develop receptor antagonists usingIL-9 dependent cell lines. In addition, this invention also providescompounds that prevent the synthesis or reduce the biologic stability ofIL-9 or the IL-9 receptor. Biologic stability is a measure of the timebetween the synthesis of the molecule and its degradation. For example,the stability of a protein, peptide or peptide mimetic⁸⁹ therapeutic maybe prolonged by using D-amino acids, or shortened by altering itssequence to make it more susceptible to enzymatic degradation.

In another embodiment, the agonists and antagonists of the invention areantibodies to IL-9 and the IL-9 receptor. The antibodies to IL-9 and itsreceptor may be either monoclonal or polyclonal made using standardtechniques well known in the art (See Harlow & Lane's Antibodies—ALaboratory Manual (Cold Spring Harbor Laboratory, 1988). They can beused to block IL-9 from binding to the receptor. In one embodiment theantibodies interact with IL-9. In another embodiment the antibodiesinteract with the IL-9 receptor. The IL-9 used to elicit theseantibodies can be any of the IL-9 varients discussed above.

Antibodies are also produced from peptide sequences of IL-9 or the IL-9receptor using standard techniques in the art (see Protocols inImmunology, Chapt. 9, Wiley). The peptide sequences from the murine IL-9receptor that can be used to produce blocking antisera have beenidentified as: GGQKAGAFTC (residues 1-10) (SEQ ID NO:19);LSNSIYRIDCHWSAPELGQESR (residues 11-32) (SEQ ID NO:20); andCESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue added to theN-terminus for coupling the peptide to the carrier protein) (SEQ IDNO:21). In addition, an epitope that binds to a blocking antibodydirected to the human IL-9 receptor has been identified as residues 8-14of the mature human IL-9 receptor. (TCLTNNI) (SEQ ID NO:22) and twoepitopes that bind to blocking antibodies directed to human IL-9 havealso been identified as residues 50-67 (CFSERLSQMTNTTMQTRY) (SEQ IDNO:23) and residues 99-116 (TAGNALTFLKSLLEIFQK) (SEQ ID NO:16) The humanepitopes as well as the human peptides that correspond to the peptidesthat produce blocking antibodies in the murine sequences are most likelyto be useful for the production of therapeutic antibodies.

In still another embodiment, the compounds of the invention may becoupled to chemical moieties, including proteins that alter thefunctions or regulation of the IL-9 pathway for therapeutic benefit inatopic allergy and asthma.⁶¹ These proteins may include in combinationother cytokines and growth factors including⁶⁷ L-4, IL-5, IL-3, IL-2,IL-13, and IL-10 that may offer additional therapeutic benefit in atopicallergy and asthma. In addition, the IL-9 of the invention may also beconjugated through phosphorylation and conjugated to biotinylate,thioate, acetylate, iodinate, and any of the crosslinking reagents shownin FIG. 27 (Pierce).

In a further embodiment, the invention includes the down regulation ofIL-9 expression or function by administering soluble IL-9 receptormolecules that bind IL-9. Renauld et al.⁵⁹ have shown the existence of asoluble form of the IL-9 receptor. This molecule can be used to preventthe binding of IL-9 to cell bound receptor and act as an antagonist ofIL-9. Soluble receptors have been used to bind cytokines or otherligands to regulate their function.⁸⁷ A soluble receptor is a form of amembrane bound receptor that occurs in solution, or outside of themembrane. Soluble receptors may occur because the segment of themolecule which commonly associates with the membrane is absent. Thissegment is commonly referred to in the art as the transmembrane domainof the gene, or membrane binding segment of the protein. Thus, in oneembodiment of the invention, a soluble receptor may represent a fragmentor an analog of a membrane bound receptor. In another embodiment of theinvention, the structure of the segment that associates with themembrane may be modified (e.g. DNA sequence polymorphism or mutation inthe gene) so the receptor is not inserted into the membrane, or thereceptor is inserted, but is not retained within the membrane. Thus, asoluble receptor, in contrast to the corresponding membrane bound form,differs in one or more segments of the gene or receptor protein that areimportant to its association with the membrane.^(52,53)

These compounds may be known forms of a soluble IL-9 receptor that actto bind IL-9. Alternatively, these compounds may resemble known forms ofthe IL-9 receptor, but may exist as fragments. In another embodiment ofthe invention, the compound may retain functions comparable to solubleIL-9 receptor, but may not resemble soluble IL-9 receptor incomposition. For example, the composition of the compound may includemolecules other than amino acids. Thus, these compounds will bind IL-9and prevent IL-9 from acting at its cell surface receptor.

A further embodiment of the invention relates to antisense or genetherapy. It is now known in the art that altered DNA molecules can betailored to provide a specific selected effect, when provided asantisense or gene therapy. The native DNA segment coding for IL-9receptor, has, as do all other mammalian DNA strands, two strands; asense strand and an antisense strand held together by hydrogen bonding.The mRNA coding for the receptor has a nucleotide sequence identical tothe sense strand, with the expected substitution of thymidine byuridine. Thus, based upon the knowledge of the receptor sequence,synthetic oligonucleotides can be synthesized. These oligonucleotidescan bind to the DNA and RNA coding for the receptor. The activefragments of the invention, which are complementary to mRNA and thecoding strand of DNA, are usually at least about 15 nucleotides, moreusually at least 20 nucleotides, preferably 30 nucleotides and morepreferably may be 50 nucleotides or more. The binding strength betweenthe sense and antisense strands is dependent upon the total hydrogenbonds. Therefore, based upon the total number of bases in the mRNA, theoptimal length of the oligonucleotide sequence may be easily calculatedby the skilled artisan.

The sequence may be complementary to any portion of the sequence of themRNA, i.e., it may be proximal to the 5′-terminus or capping site, ordownstream from the capping site, between the capping site and theinitiation codon and may cover all or only a portion of the non-codingregion or the coding region. The particular site(s) to which theantisense sequence binds will vary depending upon the degree ofinhibition desired, the uniqueness of the sequence, the stability of theantisense sequence, etc.

In the practice of the invention, expression of the IL-9 receptor isdown-regulated by administering an effective amount of syntheticantisense oligonucleotide sequences described above. The oligonucleotidecompounds of the invention bind to the mRNA coding for human IL-9 orIL-9 receptors thereby inhibiting expression (translation) of theseproteins. See Gruss et al., “Interleukin 9 is expressed by primary andcultural Hodgkin and Reed-Sternberg cells.” Cancer Res. 52:1026-31 (Feb.15, 1992)

The isolated DNA sequences containing various mutations such as pointmutations, insertions, deletions, or spliced mutations of IL-9 areuseful in gene therapy as well.

In addition to the direct regulation of the IL-9 receptor, thisinvention also encompasses methods of downstream regulation whichinvolve inhibition of signal transduction. In particular, a furtherembodiment of this invention is drawn to inhibition of tyrosinephosphorylation. It is known in the art that highly exergonicphosphoryl-transfer reactions are catalyzed by various enzymes known askinases. In other words, a kinase transfers phosphoryl groups betweenATP and a metabolite. IL-9 induces tyrosine phosphorylation of multipleproteins; it is known in the art that in addition to the activation ofJAK1 and JAK3 tyrosine kinases, IL-9 also induces tyrosinephosphorylation of Stat3.⁵⁸ Phoshorylation of Stat3 is unique to theIL-9 signal transduction pathway and hence is a perfect target forinhibitors.⁵⁸ This invention includes within its scope tyrphostins whichare specific inhibitors of protein tyrosine kinases. Thus, tyrphostins(obtained for example from Calbiochem) and other similar inhibitors ofthese kinases are useful in the modulation of signal transduction andare useful in the treatment of atopic allergies and asthma.

In still another aspect of the invention, it was surprisingly, foundthat aminosterol compounds are also useful in the inhibition of signaltransduction due to IL-9 stimulation. Aminosterol compounds which areuseful in this invention are described in U.S. patent application, Ser.No. 08/290,826 and its related applications Ser. Nos. 08/416,883 and08/478,763 as well as in Ser. No. 08/483,059 and its related applicationSer. Nos. 08/483,057, 08/479,455, 08/479,457, 08/475,572, 08/476,855,08/474,799 and 08/487,443, which are specifically incorporated herein byreference.

In addition, the invention includes pharmaceutical compositionscomprising the compounds of the invention together with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectionable solutions. Suitable pharmaceutical carriers are describedin Martin, E. W., Remington's Pharmaceutical Sciences, specificallyincorporated herein by reference.

The compounds used in the method of treatment of this invention may beadministered systemically or topically, depending on such considerationsas the condition to be treated, need for site-specific treatment,quantity of drug to be administered, and similar considerations.

Topical administration may be used. Any common topical formation such asa solution, suspension, gel, ointment, or salve and the like may beemployed. Preparation of such topical formulations as are well describedin the art of pharmaceutical formulations as exemplified, for example,by Remington's Pharmaceutical Science, Edition 17, Mack PublishingCompany, Easton, Pa. For topical application, these compounds could alsobe administered as a powder or spray, particularly in aerosol form. Theactive ingredient may be administered in pharmaceutical compositionsadapted for systemic administration. As is known, if a drug is to be aadministered systemically, it may be confected as a powder, pill,tablets or the like, or as a syrup or elixir for oral administration.For intravenous, intraperitoneal or intra-lesional administration, thecompound will be prepared as a solution or suspension capable of beingadministered by injection. In certain cases, it may be useful toformulate these compounds in suppository form or as an extended releaseformulation for deposit under the skin or intermuscular injection. In apreferred embodiment, the compounds of this invention be administered byinhalation. For inhalation therapy the compound may be in a solutionuseful for administration by metered dose inhalers, or in a formsuitable for a dry powder inhaler.

An effective amount is that amount which will down regulate either theexpression of IL-9 or the functions controlled by IL-9. A giveneffective amount will vary from condition to condition and in certaininstances may vary with the severity of the condition being treated andthe patient's susceptibility to treatment. Accordingly, a giveneffective amount will be best determined at the time and place throughroutine experimentation. However, it is anticipated that in thetreatment of asthma-related disorders in accordance with the presentinvention, a formulation containing between 0.001 and 5 percent byweight, preferably about 0.01 to 1%, will usually constitute atherapeutically effective amount. When administered systemically, anamount between 0.01 and 100 mg per kg body weight per day, butpreferably about 0.1 to 10 mg/kg, will effect a therapeutic result inmost instances.

Applicant also provides for a method to screen for the compounds thatdown regulate the expression of IL-9 or the functions controlled byIL-9. One may determine whether the functions expressed by IL-9 aredown-regulated using techniques standard in the art.⁵⁷⁻⁶⁰ In a specificembodiment, applicant provides for a method of identifying compoundswith functions comparable to Met IL-9. Thus, in one embodiment, serumtotal IgE may be measured using techniques well known in the art⁴² toassess the efficacy of a compound in down regulating the functions ofIL-9 in vivo. In another embodiment, bronchial hyperresponsiveness,bronchoalveolar lavage, and eosinophilia may be measured usingtechniques well known in the art⁴² to assess the efficacy of a compoundin down regulating the functions of IL-9 in vivo. In yet anotherembodiment, the functions of IL-9 may be assessed in vitro. As is knownto those in the art, human IL-9 specifically induces the rapid andtransient tyrosine phosphorylation of multiple proteins in M07e cells.The tyrosine phosphorylation of Stat3 transcriptional factor appears tobe specifically related to the actions of IL-9. Another method tocharacterize the function of IL-9 and IL-9-like molecules that dependson the “stable expression” of the IL-9 receptor uses the well knownmurine TS1 clones to assess human IL-9 function with a cellularproliferation assay.⁵⁹

The invention also includes a simple screening assay for saturable andspecific ligand binding based on cell lines that express the IL-9receptor.^(46,59) The IL-9 receptor is expressed in on a wide variety ofcell types, including K562, C8166-45, B cells, T cells, mast cells,neutrophils, megakaryocytes (UT-7 cells),⁵³ the human megakaryoblasticleukemia cell lines MO7e⁵⁷, TF1,⁵⁹ macrophages, fetal thymocytes, thehuman kidney cell line 293,⁵³ and murine embryonic hippocampalprogenitor cell lines.^(46,52,53) In another embodiment, soluble IL-9receptor may be used to evaluate ligand binding and potential receptorantagonists.

The practice of the present invention will employ the conventional termsand techniques of molecular biology, pharmacology, immunology, andbiochemistry that are within the ordinary skill of those in the art.See, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2d ed. Cold Spring Harbor Laboratory Press [1985], or Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.[1994].

Nonetheless, we offer the following basic background information. Thebody's genetic material, or DNA, is arranged on 46 chromosomes, whicheach comprises two arms joined by a centromere. Each chromosome isdivided into segments designated p or q. The symbol p is used toidentify the short arm of a chromosome, as measured from the centromereto the nearest telomere. The long arm of a chromosome is designated bythe symbol q. Location on a chromosome is provided by the chromosome'snumber (i.e., chromosome 5) as well as the coordinates of the p or qregion (i.e., q31-q33). In addition, the body bears the sex chromosomes,X and Y. During meiosis, the X and Y chromosomes exchange DNA sequenceinformation in areas known as the pseudoautosomal regions.

DNA, deoxyribonucleic acid, consists of two complementary strands ofnucleotides, which include the four different base compounds, adenine[A], thymine [T], cytosine [C], and guanine [G]. A of one strand bondswith T of the other strand while C of one strand bonds to G of the otherto form complementary “base pairs,” each pair having one base in eachstrand.

A sequential grouping of three nucleotides [a “codon”] codes for oneamino acid. Thus, for example, the three nucleotides CAG codes for theamino acid Glutamine. The 20 naturally occurring amino acids, and theirone letter codes, are as follows: Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Asparagine or Asx B Aspartic acidCysteine Cys C Glutamine Gln Q Glutamine Acid Glu E Glutamine or Glx ZGlutamic acid Glycine Gly G Histidine His H Isoleucine Ile I Leucine LeuL Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P SerineSer S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Amino acids comprise proteins. Amino acids may be hydrophilic, i.e.,displaying an affinity for water, or hydrophobic, i.e., having anaversion to water. Thus, the amino acids designated as G, A, V, L, I, P,F, Y, W, C and M are hydrophobic and the amino acids designated as S, Q,K, R, H, D, E, N and T are hydrophilic. In general, the hydrophilic orhydrophobic nature of amino acids affects the folding of a peptidechain, and consequently the three dimensional structure of a protein.

DNA is related to protein as follows:

Genomic DNA comprises all the DNA sequences found in an organism's cell.It is “transcribed” into messenger RNA [“mRNA”]. Complementary DNA[“cDNA”] is a complementary copy of mRNA made by reverse transcriptionof mRNA. Unlike genomic DNA, both mRNA and cDNA contain only theprotein-encoding or polypeptide-encoding regions of the DNA, theso-called “exons.” Genomic DNA may also include “introns,” which do notencode proteins.

In fact, eukaryotic genes are discontinuous with proteins encoded bythem, consisting of exons interrupted by introns. After transcriptioninto RNA, the introns are removed by splicing to generate the maturemessenger RNA (mRNA). The splice points between exons are typicallydetermined by consensus sequences that act as signals for the splicingprocess. Splicing consists of a deletion of the intron from the primaryRNA transcript and a joining or fusion of the ends of the remaining RNAon either side of the excised intron. Presence or absence of introns,the composition of introns, and number of introns per gene, may varyamong strains of the same species, and among species having the samebasic functional gene. Although in most cases, introns are assumed to benonessential and benign, their categorization is not absolute. Forexample, an intron of one gene can represent an exon of another. In somecases, alternate or different patterns of splicing can generatedifferent proteins from the same single stretch of DNA. In fact,structural features of introns and the underlying splicing mechanismsform the basis for classification of different kinds of introns.

As to the exons, these can correspond to discrete domains or motifs, asfor example, functional domains, folding regions, or structural elementsof a protein; or to short polypeptide sequences, such as reverse turns,loops, glycosylation signals and other signal sequences, or unstructuredpolypeptide linker regions. The exon modules of the presentcombinatorial method can comprise nucleic acid sequences correspondingto naturally occurring exon sequences or naturally occurring exonsequences which have been mutated (e.g. point mutations, truncations,fusions).

Returning now to the manipulation of DNA, DNA can be cut, spliced, andotherwise manipulated using “restriction enzymes” that cut DNA atcertain known sites and DNA ligases that join DNA. Such techniques arewell known to those of ordinary skill in the art, as set forth in textssuch as Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed.Cold Spring Harbor Laboratory Press [1985] or Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. [1994].

DNA of a specific size and sequence can then be inserted into a“replicon,” which is any genetic element, such as a plasmid, cosmid, orvirus, that is capable of replication under its own control. A“recombinant vector” or “expression vector” is a replicon into which aDNA segment is inserted so as to allow for expression of the DNA, i.e.,production of the protein encoded by the DNA. Expression vectors may beconstructed in the laboratory, obtained from other laboratories, orpurchased from commercial sources.

The recombinant vector [known by various terms in the art] may beintroduced into a host by a process generically known as“transformation.” Transformation means the transfer of an exogenous DNAsegment by any of a number of methods, including infection, directuptake,.transduction, F-mating, microinjection, or electroporation intoa host cell.

Unicellular host cells, known variously as recombinant host cells,cells, and cell culture, include bacteria, yeast, insect cells, plantcells, mammalian cells and human cells. In particularly preferredembodiments, the host cells include E. coli, Pseudonas, Bacillis,Streptomyces, Yeast, CHO, R1-1, B-W, LH, COS-J, COS-7, BSC1, BSC40,BMT10, and S69 cells. Yeast cells especially include Saccharomyces,Pichia, Candida, Hansenula, and Torulopis.

As those skilled in the art recognize, the expression of the DNA segmentby the host cell requires the appropriate regulatory sequences orelements. The regulatory sequences vary according to the host cellemployed, but include, for example, in prokaryotes, a promoter,ribosomal binding site, and/or a transcription termination site. Ineukaryotes, such regulatory sequences include a promoter and/or atranscription termination site. As those in the art well recognized,expression of the polypeptide may be enhanced, i.e., increased over thestandard levels, by careful selection and placement of these regulatorysequences.

In other embodiments, promoters that may be used include the humancytomegalovirus (CMV) promoter, tetracycline inducible promoter, simianvirus (SV40) promoter, moloney murine leukemia long terminal repeat(LTR) promoter, glucocorticoid inducible murine mammary tumor virus(MMTV) promoter, Herpes thymidine kinase promoter, murine and humanβ-actin promoters, HTLV1 and HIV IL-9 5′ flanking region, human andmouse IL-9 receptor 5′ flanking region, bacterial tac promoter anddrosophila heat shock scaffold attachment region (SAR) enhancerelements.

The DNA may be expressed as a polypeptide of any length such aspeptides, oligopeptides, and proteins. Polypeptides also includetranslational modifications such as glycosylations, acetylations,phosphorylations, and the like.

Another molecular biologic technique of interest to the presentinvention is “linkage analysis.” Linkage analysis is an analytic methodused to identify the chromosome or chromosomal region that correlateswith a trait or disorder.⁴⁴ Chromosomes are the basic units ofinheritance on which genes are organized. In addition to genes, artisanshave identified “DNA markers” on chromosomes. DNA markers are knownsequences of DNA whose identity and sequence can be readily determined.Linkage analysis methodology has been applied to the mapping of diseasegenes, for example, genes relating to susceptibility to asthma, tospecific chromosomes.^(42,44)

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed. It is intended that the specifications and examplesbe considered exemplary only with a true scope of the invention beingindicated by the claims.

Methods

In conducting the experiments described in the Examples below, applicantused the following methods:

Patient Populations

Asthma families were recruited from two sources.^(27,42,79-83,88) Ineach case patients were genotyped with respect to the polymorphism atposition 3365 of the human IL-9 gene [GenBank accession number M30136].

A third population of 74 individuals was ascertained randomly withrespect to asthma and atopy from the East Coast of the United States.The frequency of the Met substitution at codon 117 was used as anunbiased estimate of the prevalence of this variant in the generalpopulation.

A fourth population of 49 individuals was ascertained randomly withrespect to asthma and atopy from the Philadelphia, Pa. area. Total serumIgE were assayed by enzyme-linked immunosorbent test (ELISA, Genzyme,Cambridge, Mass.]. DNA was extracted from the WBC in peripheral bloodfrom each individual. Analyses of genetic markers (genotyping) andcandidate genes were performed on the genomic DNA extracted. Once again,the frequency of the Met substitution at codon 117 was used as anunbiased estimate of the prevalence of this variant in the generalpopulation.

Oligonucleotide Primers.

All primers were designed using OLIGO 4.0. Characterization of the IL-9gene was carried out using primers surrounding each of the 5 exons ofthe reported sequence. The primer sequences surrounding each exon were:exon 1 [upper] [5′ GCT CCA GTC CGC TGT CAA 3′] and [lower] [5′ CTC CCCCTG CAG CCT ACC 3′] [product size 150 bp); exon 2 [upper] [5′ CGG GGCTGA CTA AAG GTT CT 3′] and [lower] [5′ GTT CTT AAA GAG CAT TCA CT 3′][product size 99 bp); exon 3 [upper] [5′ ATT TTC ACA TCT GGA ATC TTC ACT3′] and [lower][5′ AAT CCA AGG TCA ACA TTA TG 3′] [product size 113 bp];exon 4 [upper] [5′ TTT CTT TGA ATA AAT CCT TAC 3′] and [lower] [5′ GAAATC ACC AAC AGG AAC ATA 3′] [product size 206 bp]; and exon 5 [upper][5′ATC AAC TTT CAT CCC CAC AGT 3′] and [lower] [5′ GGA TAA ATA ATA TTTCAT CTT CAT 3′]. Each exon was examined first by a single strandconformational polymorphism assay [SSCP].^(72,77) The primers for exon 5produced a 160 bp product after polymerase chain reaction [PCR]amplification which was also examined by direct solid phase sequenceanalysis.^(72,77) The upper primer was synthesized with a 5′ biotinlabel and, following amplification, the PCR product was captured by astreptavidin-linked paramagnetic bead [Dynal] and characterized bySanger sequencing as described elsewhere.⁷⁷ Sequence polymorphisms weredistinguished from artifact by repeated analyses.

SSCP Analysis.

SSCP, a method for detection of polymorphisms on the basis of changes inmigration of single-stranded DNA exposed to an electric field,⁷² wascarried out as set forth in Schwengel et al., (1994) at room temperaturewith and without 10% glycerol using 6% polyacrylamide gelelectrophoresis at a cross-linking monomer concentration of 2.67%.⁷⁷Four μl of PCR product was mixed with 5 μl 2× stop buffer [95%formamide, 20 mM EDTA, 0.05% BPB, 0.05% xylene cyanol), and 1 μl 0.5%SDS and 50 μM EDTA, denatured at 85-90° C. for 8 minutes, and thenimmediately placed on ice. Electrophoresis was carried out at 12 wattsfor approximately 24 hours for glycerol containing gels and 12 hours fornon-glycerol gels. The gels were then dried and exposed to Kodak XAR®film.

DNA Sequencing.

Direct DNA sequencing of the PCR products was accomplished using solidphase techniques after verifying the presence of the correct size PCRproduct on a 1% agarose gel stained with ethidium bromide as set forthin Schwengel et al., 1994.⁷⁷ Twenty μl of PCR product was incubated with40 μl of Dynabeads® m-280 [Dynal] for 15 minutes. The beads were washedand diluted as suggested by the manufacturer. Each sample wassubsequently washed with B&W buffer containing 10 mM tris-HCl pH 7.5, 1mM EDTA, 2 M NaCl, denatured with 0.1 N NaOH, and then washed with 0.1 NNaOH, B&W buffer, and 10 mM Tris-HCl pH 8 and 1 mM EDTA [TE]. The pelletof beads was resuspended with 10 μl of H2O.

Sanger sequencing reactions were carried out using Sequenase (UnitedStates Biochemical Co.]. ³⁵S-dATP or ³³P-dATP was incorporated into thesequencing reactions, and the products were electrophoresed througheither 5% or 6% polyacrylamide gels containing 7 M urea. Gels were driedwithout fixing and exposed to X-ray film. Alleles were determined bycomparing the genotypes of parents and offspring. Infrequent artifactswere easily distinguished from true sequence polymorphisms byrepetition.

DNA was available and extracted from peripheral leukocytes. Genomic DNAwas diluted to a concentration of 200 μg/ml for amplification.^(27,42)Simple sequence repeats [SSR] including DXYS154 were selected from theGenome Data Base [GDB; Welch library, Johns Hopkins University,Baltimore, Md.]. Genotyping of the sKK-1 marker was carried out usingthe following primers sKK-1U (5′ CAA ATC TGA AGA GCA AAC TAT 3′] andsKK-1L [5′ TTA AAA AAT TCA TTT CAG TAT TCT 3′] which produce a 90 bpproduct. Each SSR product was amplified by PCR⁷² and sized according tomethods previously described.^(27,42) Sample handling was carried out asdescribed by Weber et al. with minor modifications.^(71,27,42) Genotypeswere determined from two independent readings of each autoradiograph.Individuals genotyping the families were blinded to the clinical data.

RFLP Analysis.

As a result of the C to T polymorphism at position 3365, a StyIrestriction fragment length polymorphism [RFLP] was produced at position52 of the IL-9 exon 5 PCR product. To test for the presence of this DNAsequence variant the lower primer from exon 5 was end-labeled prior toPCR amplification. The PCR product was then digested with StyI producingtwo fragments 108 bp [labeled] and 52 bp [unlabeled] in length. ThisRFLP was used along with SSCP to confirm the presence of thispolymorphism in families and individuals.

Linkage Analyses and Data Management.

Linkage analyses were performed using affected sib-pair methods [SIBPAL,S.A.G.E.],⁷⁸ an established approach for the investigation of thegenetic basis of complex traits, such as BHR, atopy, and asthma.Affected sib-pairs are usually tested first, since a proportion ofunaffected sib-pairs may still be gene carriers but do not express thetrait. In contrast to LOD score methods where the model of inheritance[dominant, recessive, etc.] must be specified exactly, analysis bysib-pair methods makes no explicit assumptions in this regard. Thus, insib-pair analyses the parents' clinical information is not used intesting for linkage. The pertinent observation in these methods is howoften two affected offspring share copies of the same parental markerallele.⁴⁴ If the same copy of a parental marker allele is observed indifferent offspring, they are said to be inherited “identical bydescent.” Linkage is suggested when affected sib-pairs are identical bydescent for a marker allele significantly more often than expected bychance [50%]. When the same marker allele is transmitted with thedisease gene in different offspring, this implies that the marker locusis linked, or must be located close enough on the same chromosome, tothe disease gene so they cosegregate during meiosis. The trait is thenmapped by knowing the chromosomal localization of the marker.

Linkage in humans may also be established by the method of likelihoodratios. This method involves comparison of the probability that observedfamily data would arise under one hypothesis, for instance, linkagebetween two DNA markers, to the probability that it would arise under analternative hypothesis, typically, nonlinkage. The ratio of theseprobabilities is called the odds ratio for one hypothesis relative tothe other. By convention, mammalian geneticists prefer the log of theodds ration, or the LOD score. Generally, linkage is considered provedwhen the odds in favor of linkage versus nonlinkage become overwhelming,or reach 1,000:1 [LOD=3]. Linkage is rejected when the odds drop to100:1 against this hypothesis [LOD=−2]. The maximum likelihood estimateis the recombination fraction where the likelihood ratio is largest.LODs from multiple pedigrees are thus added until the score grows to 3[signifying 1,000:1 odds] or falls to −2 [indicating 100:1 odds].

All clinical and genotype data is managed using EXCELL® on a MacIntosh®or Sun Microsystems® computer. Statistical analyses were preformed usingJMP [SAS Institute, Inc. Cary, N.C.]. The Wilcoxon/Kruskal-Wallis Tests[rank sums] was used to test whether individuals who were homozygous[Met/Met], heterozygous [Met/Thr], or homozygous [Thr/Thr] at codon 117differ in their serum total IgE. All P-values are two-tailed exceptaffected sib-pair analyses, where a one-tailed test was used becauseonly an increased sharing of alleles was expected.

Having provided this background information, applicant now describespreferred aspects of the invention.

EXAMPLE 1 Linkage Analysis Between BHR and Murine Chromosome 13

As an aid in dissecting the complex genetic determinants of BHR,applicant has developed murine models that differ in their geneticsusceptibility to various bronchoconstrictor stimuli. Inbred animalmodels using recombinant inbred strains [BXD] can facilitate ongoingstudies in humans to determine the number of genes regulatingsusceptibility to BHR, the magnitude of their affect, and their precisechromosomal location. In particular, localizing in an animal model agene determining susceptibility to a critical risk factor for asthma mayaid in the positional cloning of this gene in humans.

Although the gene[s] predisposing to BHR and atopy had not yet beenidentified prior to this invention, chromosome 5q31-q33 was known to besyntenic with portions of mouse chromosomes 11, 13, and 18. FIG. 2illustrates the syntenic regions containing numerous positionalcandidates that may potentially play a role in airway inflammationassociated with BHR, atopy, and asthma. Specifically, the region ofhuman chromosome 5q31-q33 demonstrating significant evidence for linkagewith BHR is homologous to portions of mouse chromosomes 11, 13, and 18which contain numerous candidate genes.⁸⁴

In particular, IL-9 or a nearby gene have recently been suggested aslikely candidates on the basis of linkage disequilibrium between logserum total IgE levels and a marker in this gene using a randomlyascertained population.⁴³

Despite comparisons with four candidate intervals, evidence for linkagewas found for only one region, designated Aib 1 [atracurium inducedbronchoconstriction 1]. FIG. 3 provides the results. Specifically, FIG.3 sets forth the LOD score curve on mouse chromosome 13 foratracurium-induced airway responsiveness in 24 BXD RI strains which arederived from the hyporesponsive C57BL/6J and the hyperresponsive DBA/2Jprogenitor strains [solid line]. The LOD score curve resulting from theselective genotyping of 20 BXD strains is also shown [dashed line]. BXDstrains -2, -6, -18, and -32 were not used in the second analysis sincethey were intermediate in phenotype displaying a mean response greaterthan 1 standard deviation below the DBA/2 and above the C57BL/6 meanresponses. The bronchoconstrictor response to atracurium, 20 mg/kg givenintravenously, was assessed by the change in peak inspiratory pressureintegrated over time [4 min], termed the airway pressure time index[APTI]. Atracurium-induced APTI was measured in 2-8 animals per RIstrain. Marker data were obtained from the RWE data base in the MapManager data analysis program. The genetic distance [cM] between markersis indicated on the abscissa. LOD scores were calculated by theMAPMAKER/QTL linkage program. A QTL was detected in this region andtermed atracurium-induced bronchoconstriction-1 [Aib1].

FIG. 3 indicates that this quantitative trait locus [QTL] is located onthe midportion of murine chromosome 13 and attained this interval amaximum likelihood log of the odds [LOD] of 2.42. Forty-four percent ofthe total variance in atracurium-induced bronchoconstriction wasexplained at Aib1 when all of the markers in the BXD map were analyzed.The LOD for chromosome 13 increased to 2.85 when QTL analyses were runafter excluding the four strains [BXD-2, -6, -18, and -32] that wereintermediate responders to atracurium. The known positional candidatesin the linked region of chromosome 13 include: D1 dopamine receptor[Drd1], fibroblast growth factor receptor 4 [Fgfr4], lymphocyteantigen-28 [Ly28], thiopurine methyltransferase [Tpmt], and IL-9.

Because the applicant was specifically testing for linkage to fourcandidate regions in the mouse, based on previous mapping data in thehuman, the data presented here are highly significant. As stated in theclassic paper by Lander and Botstein,⁶⁷ a false positive rate forlinkage will result if the LOD threshold (T) is chosen so that T=½(log10 e)(Z α/n)2 (where n is equal to the number of intervals tested).Typically a minimum LOD of 3.3 is required as evidence of linkage.⁶⁷However, this threshold is based on the assumption that one is searchingthe entire genome. These same authors point out that a LOD of 0.83 issufficient when only one region is examined. In this case, with fourcandidate regions, a P value (α) of 0.0125 for each region is requiredto obtain a true P≦0.05, when one corrects for multiple independentcomparisons. Adopting a 5% error rate that even a single false positivefinding will occur, as suggested by Soller and Brody,⁶⁸ and solving theequation ½(log 10 e)(Z α/n)², yields a LOD threshold of at least 1.36. Amaximal LOD of 1.48 was obtained for the Il-9 gene candidate.Restricting the acceptable false positive error rate to ≦0.1%, increasesthe LOD threshold to 2.36. Thus, the maximal LOD generated of 2.42 forthe candidate interval on chromosome 13 (Aib1) is highly significant.

These LOD threshold data provide evidence of a conserved linkage for BHRin humans and mice. BHR in humans links to the region on chromosome 5qcontaining a number of growth factors and cytokines including the IL-9gene and the Aib1 locus maps to the IL-9 region of murine chromosome 13.

EXAMPLE 2 Identification of an IL-9 Gene Polymorphism

Applicant demonstrated conserved linkage between the mouse and humansfor BHR. These data suggest that variation in the functions of this geneor DNA sequence may be important in regulating bronchial responsivenessin the mouse. Using the methods described above, a unique product of thecorrect size was identified by gel electrophoresis for each of the exonsof human IL-9 after PCR. A single polymorphism was identified by SSCP inexon 5 of the human IL-9 gene. Direct DNA sequence analysis demonstrateda C to T nucleotide substitution at position 3365 [GenBank accessionnumber M30136] of the human IL-9 gene as the cause of the novel SSCPconformer. This DNA sequence change predicts a nonconservativesubstitution of a methionine [hydrophobic] for a threonine [hydrophilic]at amino acid 117 of the IL-9 protein.

Exon 5 codes for this segment of the protein which is within the mosthighly conserved interval of human IL-9 as compared to the mouse IL-9sequence (see FIG. 4).

Individuals were genotyped from various populations to examine thefrequency of these alleles by direct analyses of the nucleotidesubstitution in the coding sequence of human IL-9. Two of 394individuals from a group of asthmatic families were homozygous [Met/Met]at codon 117 [0.5%]. There were 91 [23.1%] heterozygous, and 301 [76.4%]homozygous [Thr/Thr] individuals. The true prevalence for this IL-9variant is likely to be significantly higher because the Italianpopulation of families was ascertained through symptomatic patients withasthma. From a separate ethnically diverse population ascertainedrandomly with respect to atopy and asthma, there were 1 of 49individuals homozygous for [Met/Met] at codon 117 [2.0%]. There were 11[22.4%] heterozygous, and 37 [75.5%] homozygous [Thr/Thr] individuals.The prevalence of the Met/Thr heterozygotes was 18.9% in a fourthpopulation ascertained randomly with respect to atopy and asthma. Thus,approximately 20% of the population are likely to represent carriers ofthe T allele at position 3,365 as compared to the reported sequence[GenBank accession number M30136]. Because it is well known in the artthat the frequency of any allele in the population is p2+2 pq+q2, then,approximately 4% of the population is expected to be Met/Met homozygousat codon 117 of IL-9.

Overall, serum total IgE averaged 44.5 I.U. for homozygous individuals[Met/Met], which was significantly different from those who werehomozygous wild type [Thr/Thr] [351.7I.U.], or heterozygous [Met/Thr][320.9 I.U.]. See FIG. 5. The homozygous protected individuals [Met/Met]failed to demonstrate evidence of atopic allergy except for a singlepositive skin test in one individual. These data indicate that thisnovel DNA polymorphism, when inherited in the homozygous state, isassociated with protection from atopic allergy, including lower serumtotal IgE.

FIG. 6 illustrates the PCR amplification of the IL-9 simple sequencerepeat polymorphism. This marker is compared with genotype for theseindividuals for the restriction fragment length polymorphism produced bythe nucleotide polymorphism at position 3,365 as compared to thereported sequence [GenBank accession number M30136]. Two families areshown. The individuals in lanes 1 and 2 are the parents (Thr/Met) ofindividuals in lanes 3 (Met/Thr) and 4 (Met/Met); lanes 5 (Thr/Thr) and6 (Met/Met) are parents for offspring in lanes 7 (Met/Thr) and 8(Met/Thr). The smallest allele for the IL-9 simple sequence repeatpolymorphism (the lowest band in each figure is 248 nucleotides inlength) is in complete linkage disequilibrium with the Met117 allele(nucleotide substitution at position 3,365 as compared to the reportedsequence [GenBank accession number M30136]) in these individuals and inall individuals from populations tested world wide. This was true inboth the Italian and all random ascertained ethnically diverseindividuals studied, and therefore, this marker may be useddiagnostically to detect the presence of the Met117 allele. These dataare most consistent with the hypothesis that this variant is widelydistributed in populations worldwide and arose before many of thesepopulations separated.

EXAMPLE 3 IL-9 Receptor Expression and Ligand Binding Assay

Purified recombinant Thr IL-9, Met IL-9, and compounds potentiallyresembling Met IL-9 in structure or function are radiolabelled using theBolton and Hunter reagent as described in Bolton A E, and Hunter W M,Biochem J. 133:529-539(1973). This material is labeled to high specificactivity of 2,300 cpm/fmol or greater. Human K562 and MO7e cells aregrown and resuspended at 30° C. in 0.8 ml of Dulbecco's modified Eagle'smedium supplemented with 10% (vol/vol) fetal bovine serum, 50 mM2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mML-glutamine. K562 or MO7e cells are used as is or after transfectionwith the IL-9 receptor gene as described below. Plasmid DNA containingthe full length IL-9 receptor is cloned into pRC/RSV plasmid (InVitrogen, San Diego) and purified by centrifugation through CsCl2.Plasmid DNA (50 micrograms) is added to the cells in 0.4 cm cuvettesjust before electroporation. After a double electric pulse (750 V/74ohms/40 microFaradays and 100 V/74 ohms/2100 microFaradays) the cellsare immediately diluted in fresh medium supplemented with IL-9. After 24h the cells are washed and incubated in G418 (2.5 mg/ml, GIBCO) witheither no ligand, or various concentrations of 125I-labeled ligand at20° C. for 3 h. An excess of unlabeled ligand is used in parallelexperiments to estimate nonspecific binding. The cells are then washed,filtered, and collected for counting. Specific incorporation iscalculated by Scatchard analysis. Similar competitive assays are runusing 125I-labeled Thr117 IL-9 and various amounts of putative coldligands to assess specific binding.

Soluble IL-9 receptor including amino acids 44 to 270 (R&D Systems) wasincubated with different forms of human recombinant IL-9. Varyingamounts of FlagMet117 and FlagThr117 (described in Example 7) wereincubated in PBS at room temperature for 30 minutes with 0.5 μgs ofsoluble receptor. EBC buffer (50 mM Tris pH 7.5; 0.1 M NaCl; 0.5% NP40)was added (300 μl) was added along with 1 μg of anti-FLAG monoclonalantibody (IBI) and incubated for 1 hour on ice. Forty microlitres ofprotein A sepharose solution was added to each sample and mixed for 1hour at 4° C. Samples were centrifuged for 1 minute 11,000×G and pelletswere washed 4 times with 500 μl of EBC. Pellets were dissolved in 26 μlof 2×SDS buffer, boiled for 4 minutes, and electrophoresed through an18% SDS polyacrylamide gel. Western blots were performed as described inExample 15 except the blots were probed with an anti-IL-9 receptorantibody (R&D Systems).

FIG. 25 demonstrates the binding of the IL-9 recombinant proteinssoluble IL-9 receptor. Lane 1 is molecular weight markers, lane 2 is theIL-9 FlagMet117 incubated with the receptor, lane 3 is the IL-9FlagThr117 incubated with the receptor. These data demonstrate that bothforms of the recombinant IL-9 protein are bound to the soluble receptor.Moreover, these data along with those of Example 2 (where hetrozygotesdo not differ in serum Ig-E from homozygous Thr117 individuals) areconsistent with the IL-9 Met117 form representing a weak agonist.

EXAMPLE 4 IL-9 Receptor Expression and Ligand Functional Assay in K562,C8166-45, and MO7e Cells

Recombinant Thr117 IL-9, Met117 IL-9, and compounds potentiallyresembling Met IL-9 in structure or function were purified and preparedfor use in Dulbecco's modified Eagle's medium. K562, C8166-45 or MO7ecells are used as is or after transfection with the IL-9 receptor geneas described in Example 3. After 24 h of deprivation from growth factorsthe cells are incubated without (control) or with variable amounts ofpurified Thr117 IL-9, Met117 IL-9, and compounds potentially resemblingMet117 IL-9 in structure or function. Cellular proliferation is assessedby measuring acid phosphotase activity. Briefly, quadruplicate samplesof MO7e cells are cultured in flat-bottom microtiter plates (150 or 200microliter wells) with or without ligand for 72 to 96 hours at 37degrees C. Acid phosphatase is measured as suggested by the manufacturer(Clontech, Palo Alto, Calif.). All experiments are repeated at leasttwice.

EXAMPLE 5 Cell Isolation and Culture

Human peripheral blood mononuclear cells {PBMC} were isolated fromhealthy donors by density gradient centrifugation using endotoxin testedFicoll-Paque PLUS according to the manufacturer (Pharmacia Biotech, ABUppsala Sweden). PBMC (5×10⁶), mouse spleen cells (5×10⁶), or 5×10⁶ MO7ecells were cultured in 7 ml of RPMI-1640 (Bethesda Research Labs (BRL),Bethesda, Md.) supplemented to a final concentration of 10% with eitherisogenic human serum or heat-inactivated FBS. Cells were cultured for 24hrs at 37° C. either unstimulated, or stimulated with either PMA 5μg/ml/PHA 5 μg/ml, or PHA 5 μg/ml/rhIL2 50U (R&D Systems, Minneapolis,Minn.).

EXAMPLE 6 RN, Isolations. RT-PCR, Cloning and Sequencing of RT-PCRProducts

Total cellular RNA was extracted after 24 hrs from cultured PBMC, mousespleen cells, and MO7e cells using RNA PCR corekit (Perkin-Elmer Corp,Foster City, Calif.) according to the supplier. One μg of RNA from eachsource was denatured for 5 minutes at 65° C. and then reversetranscribed into cDNA using a 20 μl reaction mixture (RNA PCR corekit,Perkin-elmer Corp, Foster City, Calif.) containing 50 U of MuMLV ReverseTranscriptase, 1 U/μl RNAse Inhibitor, 2.5 mM oligo d(T) 16 primer, 1 mMeach of dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCL, pH 7.0, 25 mMMgCl2. The reaction mixture was pipetted into thermocycler tubes, placedin a PCR thermal cycler and subjected to 1 cycle (15 minutes at 42° C.,5 minutes at 99° C., and 5 minutes at 4° C.). A mock reversetranscription reaction was used as a negative control.

Then this mixture was added to a second tube containing 2 mM MgCl2, 50mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5 μl of DI water, 2.5 U Amplitaq DNApolymerase, and 1 μl (20 μM) each of oligonucleotides representing humancDNA IL-9 exon 1 (forward) and exon 5 (reverse), for a final volume of100 μl. The reaction mixture was subjected to the following PCRconditions: 120 seconds at 98° C., then 30 cycles at: 30 seconds at 94°C.; 40 seconds at 55° C.; 40 seconds at 72° C. Finally, the reactionmixture was cycled one time for 15 minutes at 72° C. for extension.

PCR products representing hIL-9 cDNA were subjected to gelelectrophoresis through 1.5% agarose gels and visualized using ethidiumbromide staining. Products of a mock reverse transcriptase reaction, inwhich H₂O was substituted for RNA, and used as negative controlamplification in all experiments.

The PCR oligonucleotide primer pairs used in these experiments toamplify cDNA include: human interleukin 9 (hIL-9) exon 1 forward 5′-TCTCGA GCA GGG GTG TCC AAC CTT GGC G-3′ (SEQ ID NO: 1) and exon 5 reverse5′GCA GCT GGG ATA AAT AAT ATT TCA TCT TCA T-3′ (SEQ ID NO: 2); mouseinterleukin 9 (mIL-9) exon 1 forward 5′-TCT CGA GCA GAG ATG CAG CAC CACATG GGG C-3′ (SEQ ID NO: 3) and mouse exon 5 reverse 5′-GCA GCT GGT AACAGT TAT GGA GGG GAG GTT T-3′ (SEQ ID NO: 4); XhoI and PvuII restrictionenzyme recognition sequences are underlined in the human and mouse IL-9primers. PCR products were subcloned into the TA Cloning vector(Invitrogen, San Diego, Calif.). Amplification of the mouse cDNA gave a438 bp product and amplification of the human cDNA gave a 410 bpproduct.

Complementary DNAs for human IL-9 and murine mIL-9 were generated andamplified by RT-PCR using IL-9 exon 1 and 5 specific primers containingdigestion sites for XhoI and PvuII restriction endonucleases.Amplification products for hIL-9 and mIL-9 were isolated from 2.5%agarose gels using silica(Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual Cold Spring Harbor Laboratory Press, New York)(incorporated herein by reference in its entirety). After recovery, thecDNA products were ligated into the TA Cloning vector (Invitrogen Corp.,San Diego, Calif.) and then used to transform INVαF′ competent cells,according to the manufacturer's instructions. Plasmids containing hIL-9and mIL-9 cDNA inserts were isolated by conventional techniques(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory Press, New York). After amplification the DNAsequence including and surrounding each insert was analyzed forPCR-induced or cloning-induced errors.

hIL-9 and mIL-9 cDNA inserts were sequenced by the dideoxy-mediatedchain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci.USA 74:5463), using the M13 (-20) forward primer (5′-GTA AAA CGA CGG CCAGT-3′) (SEQ ID NO: 17) and Sequenase™ (USB), and analyzed by gelelectrophoresis (Sambrook, J. et al. (1989) Molecular cloning: alaboratory manual Cold Spring Harbor Laboratory Press, New York). hIL-9and mIL-9 cDNA inserts without cloning and/or Taq polymerase-inducedsequence errors (see translated cDNA sequences FIGS. 7 and 8) weresubcloned into expression vectors (see FIGS. 9-12) or used to createmissense mutations and deletion mutants.

EXAMPLE 7 Cloning and Expression of IL-9 Constructs in Vitro

General Cloning Methods for Constructs

hIL-9 was subcloned into procaryotic expression vectors. The TA2AAF1 metand thr vectors were digested by EcoRI and the 0.420 kB fragment(containing an XhoI site at the 5′ end of the hIL9 cDNA) was cloned intothe EcoRI site contained with the polylinker of pBluescript (PBS)(Stratagene). Clones in the sense orientation to the T3 promoter werethen digested with XhoI (the fragment contained a 5′ XhoI site from theIL-9 cDNA insert from TA vectors and a 3′ XhoI site from the PBSpolylinker) and inserts were subcloned into the XhoI sites of theprocaryotic expression vectors pGEX and pFLAG.

Cloning and Expression of IL-9 Constructs in the pGEX-4T-1 GlutathioneS-Transferase Gene Fusion Vector

For the expression, purification, and detection of IL-9 protein, IL-9cDNA inserts were subcloned into the XhoI site within the multiplecloning cassette of the 4.9 Kb pGEX-4T-1 glutathione s-transferase genefusion vector (Pharmacia Biotech, Piscataway, N.J.) by standardtechniques. Briefly, TA clones containing intact IL-9 cDNA sequences,and the pGEX-4T-1 vector were digested for one hour at 37° C. using XhoIand PvuII restriction endonuclease in the presence of 1× React 2 buffer(New England Biolabs, Beverly, Mass.) (total volume 50 μl). Productswere electrophoresed in a 1.5% preparative agarose gel with 10 μg/mlethidium bromide. The appropriate sized DNA band was excised, theagarose was melted at 45° C. for 10 minutes in 3 volumes of NaI stocksolution. A silica matrix solution in DI H₂O (Geneclean II, LaJolla,Calif.) was added to the solution at 5 μl per 5 μg of DNA and 1 μl per0.5 μg of DNA above 5 μg. The slurry was incubated at 4° C. andoccasionally shaken during 30 minutes. The slurry was then pelleted viamicrocentrifugation, washed 3 times in low-salt buffer and resuspendedin 10 μl of DI H₂O to elute the DNA from the silica. A finalmicrocentrifugation provided the 10 μl solution containing purified DNA.

Products were resuspended in 50 μl of DI H₂O and precipitated by theaddition of 2 volumes of ethanol and 1/10 volume 3M sodium acetate.Samples were centrifuged at RT at 14,000 rpm for 10 minutes, air driedunder negative pressure and resuspended in an appropriate volume of DIH₂O. Ligations and transformations of DH5a bacteria (GIBCO/BRL,Gaithersburg, Md.) with mIL-9 and hIL-9 cDNA inserts in the pGEX-4T-1vector were performed using standard techniques.

To confirm that the hIL-9 cDNA inserts contained in the pGEX-4T-1 vectorwere of the correct nucleotide sequence, plasmids containing candidateIL-9 cDNA were sequenced via the dideoxy-mediated chain terminationmethod using the aforementioned mIL-9 and hIL-9 cDNA-specificoligonucleotides (exon 1 forward, exon 5 reverse primers).

Recombinant fusion proteins were obtained from large scale cultures. Theovernight culture of transformed E. coli (50 ml) was inoculated intofresh LB/amp broth. The culture was incubated for 4 hr at 37° C. withvigorous shaking, isopropyl β-D-thiogalactopyranoside was then added toa final concentration of 1 mM, and the culture was incubated for anadditional 1.5 h. The cells were harvested by centrifugation at 500×g at4° C. and recombinant variants were purified by making use of affinitychromatography on glutathione-sepharose 4B column (Pharmacia) forGST-fusion proteins. Some variants were expressed as inclusion bodiesand were purified from insoluble inclusion bodies by the proceduredescribed by Marston (1987 The purification of eukaryotic polypeptidesexpressed in E. coli in Clover D. M. ed. DNA cloning: A practicalapproach, IRL Press, Oxford, 59-88). Briefly, the cells were lysed withlysozyme followed by treatment with deoxycholic acid. Contaminatingnucleic acids were removed by treatment with DNase I. The insolublematerial was washed once with 2 M urea and finally solubilized in lysisbuffer (50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 100 mM NaCl) containing 8 Murea. The solubilized components from the inclusion bodies were dialyzedstepwise against decreasing concentrations of urea (starting with 8, 6,4 and 2 M of urea) in lysis buffer to allow for refolding of thedenatured protein. Finally, the sample was dialyzed against 2 M urea and2.5% β-mercaptoethanol (β-ME) and centrifuged at 10,000 g for 15 min.The fusion protein was finally dialyzed against 0.01 M Tris-Cl, pH 8.0.Fusion proteins expressed in pGEX-4T vector were cleaved with 100 U ofThrombin for 6 hr at 37° C. and recovered in flow through fractionsafter chromatography on glutathione-Sepharose 4B column. Finalpurification was achieved by chromatography on Sephadex G-100 column(100×1.5 cm), packed and equiliberated with 0.05 M ammonium bicarbonatebuffer.

Cloning and Expression of IL-9 Constructs in the pFLAG-1™ ExpressionVector

For the expression, purification and detection of human IL-9 protein,IL-9 cDNA inserts were subcloned into the Xho2 site of the multiplecloning site (XhoI) of the 5.37 Kb Flag vector. FLAG technology iscentered on the fusion of a low molecular weight (1 kD), hydrophilic,FLAG marker peptide to the N-Terminus of a recombinant protein expressedby the pFLAG-1™ Expression Vector (1) (obtained from IBI Kodak). Eachbacterial colony was grown in LB broth containing 50 μg ampicillin perml until the optical density at 590 nm reached 0.6. IPTG was then addedto a final concentration of 1 mM, and the cultures incubated for anadditional 1 hr to induce protein synthesis. The cells were harvested bycentrifugation, and the cell pellet was boiled in 50 μl of Laemmlibuffer (Laemmli, 1970) for 10 min and electrophoresed on 10%polyacrylamide gels. The Anti-FLAG™ M1 monoclonal antibody was used forspecific and efficient detection of the FLAG fusion protein on western(see FIG. 13) slot or dot blots throughout its expression, affinitypurification, and FLAG marker removal. The FLAG fusion protein wasrapidly purified under mild, non-denaturing conditions in a single stepby affinity chromatography with the murine Anti-FLAG™ IgG M1 monoclonalantibody covalently attached to agarose. Following affinity purificationthe fusion protein may be used after removal from the affinity column orthe authentic protein may be recovered in biologically active form byspecific and efficient proteolytic removal of the FLAG peptide withenterokinase. Final purification was achieved by chromatography onSephadex G-100 column (100×1.5 cm), packed and equiliberated with 0.05 Mammonium bicarbonate buffer. The promoters described above in thisexample may also be used with FLAG technology.

SDS-PAGE and Immnunoblot Analysis

SDS-PAGE was performed by the method of Laemmli (Laemmli U.K. (1970)Nature 227, 680-685)(incorporated herein by reference in its entirety)by using a 12.5% polyacrylamide gel in a mini-gel system (SE 280vertical gel unit, Hoefer). For immunoblot analysis, the proteinsseparated by SDS-PAGE were transferred to nitrocellulose membranes byusing the TE 22 Mighty small transfer unit (Hoefer) in 25 mMTris-glycine buffer, pH 8.3, containing 15% methanol (Towbin H., et al.,(1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354). The unoccupiedbinding sites on the membrane were blocked by incubating for 1 h with 20mM Tris-HCl buffer, pH 8.0, containing 2% bovine serum albumin. Themembranes were then incubated with 1:200 dilution of antibodiesovernight at 4° C. The membranes were washed and treated with 1:2000diluted goat anti-rabbit IgG conjugated with either peroxidase oralkaline phosphotase for 1 h. After washing, the bound antibodies werevisualized by addition of the super-substrate chemiluminescent reagent(Pierce) or the 4-chloro-1-naphthol color developing reagent. Thereaction was stopped by immersing the membranes in distilled water. FIG.13 demonstrates that the purified recombinant human FLAG IL-9 fusionproteins (Met117 and Thr117) are the correct size and in the correctreading frame because they are recognized by the Anti-FLAG™ M1monoclonal antibody.

Analytical Methods

The molecular mass of the purified proteins was confirmed by matrixassisted laser desorption mass spectrometry using Perceptive BiosystemsVoyager Biospectrometry workstation. Amino acid analyses were performedafter hydrolysis of the sample in 6N HCl at 110-C for 24 h in evacuatedsealed glass bulbs.

Automated Edman Degradation

The partial amino acid sequence of the purified proteins is determinedby automated step-wise sequencing on an Applied Biosystems model 477Agas-phase sequencer with an on-line model 20A PTH analyzer.

EXAMPLE 8 Deletion of Exon 2 and/or Exon 3: Mutagenesis and Sequencingof Constructs

Human exon 2 and exon 3 deletions are created using ExSite PCR-basedsite-directed mutagenesis kit as suggested by the manufacturer(Stratagene, La Jolla, Calif.). The PCR primers are as follows: h9CD1Uforward 5′-GTG ACC AGT TGT CTC TGT TTG-3′ (SEQ ID NO: 5); h9CD1L reverse5′-CTG CAT CTT GTT GAT GAG GAA-3′ (SEQ ID NO: 6); h9CD2U forward 5′-GACAAC TGC ACC AGA CCA TGC-3′ (SEQ ID NO: 7); h9CD2L reverse 5′-ATT AGC ACTGCA GTG GCA CTT-3′ (SEQ ID NO: 8). Exon 2 deletions are created by usingthe primer pair h9CD1L forward and h9CD1L reverse. Exon 3 deletions arecreated by using h9CD2U forward and h9CD2L reverse. Deletions thatincluded exon 2 and exon 3 use the primer pair h9CD2U forward h9CD1Lreverse.

Mouse exon 2 and exon 3 deletions are created using ExSite PCR-basedsite-directed mutagenesis kit as suggested by the manufacturer(Stratagene, La Jolla, Calif.). The PCR primers are as follows: m9CD1Uforward 5′-GTG ACC AGC TGC TTG TGT CTC-3′ (SEQ ID NO: 9); m9CD1L reverse5′-CTT CAG ATT TTC AAT AAG GTA-3′ (SEQ ID NO: 10); m9CD2U forward 5′-GATGAT TGT ACC ACA CCG TGC-3′ (SEQ ID NO: 11); m9CD2L reverse 5′-GTT GCCGCT GCA GCT ACA TTT-3′ (SEQ ID NO: 12). Exon 2 deletions are created byusing the primer pair m9CD1U forward and m9CD1L reverse. Exon 3deletions are created by using m9CD2U forward and m9CD2L reverse.Deletions that included exon 2 and exon 3 use the primer pair m9CD2Uforward m9CD1L reverse.

Mutagenized constructs of the hIL-9 and mIL-9 cDNA inserts are sequencedby the dideoxy-mediated chain termination method (Sanger et al. (1977)Proc. Natl. Acad. Sci. USA 74:5463) (incorporated herein by reference inits entirety), using the M13 (-20) forward primer(5′-GTAAAACGACGGCCAGT-3′) (SEQ ID NO: 18) and Sequenase™ (USB), withanalysis by gel electrophoresis (Sambrook, J. et al. (1989) Molecularcloning: a laboratory manual Cold Spring Harbor Laboratory Press, NewYork). Mutants that lack exon 2, exon 3, or both exon 2 and exon 3, andare without Taq polymerase-induced sequence errors can be used to createexpression vectors.

EXAMPLE 9 Cell Lines, Cellular Proliferation Assays and Inhibition ofIL-9 Activity

Cell lines were used to assess the function of peptides, aminosterols,tyrophostins, rhIL-9, rmIL-9, and recombinant mutant forms of theseproteins as well as all other compounds that block IL-9 function. Aproliferative response was measured and compared to each of the othercytokines, variant or mutant forms of Il-9, or IL-9 antagonists. Inaddition, compounds were tested for their ability to antagonize thebaseline proliferative response. Once a baseline proliferative responsewas established for a cytokine a statistically significant loss ofresponse in assays repeated three times in triplicate was consideredevidence for antagonism. A true antagonistic response was differentiatedfrom cellular toxicity by direct observation, trypan blue staining (atechnique well known to one of normal skill in the art), and loss ofacid phosphatase activity. Specificity was assessed for the antagonistby evaluating whether the activity was substantially expressed againstother proliferative agents such as steel factor, interleukin 3, orinterleukin 4.

The MO7e line is a human megakaryoblastic cell line, cultured in RPMI1640 (GIBCO/BRL, Gaithersburg, Md.), 20% Fetal Bovine Serum (Hyclone)and 10 ng/ml IL-3 (R&D Systems, Minneapolis, Minn.). The MJ line is acytokine independent human lymphoblastoid cell line grown in RPMI 1640(GIBCO/BRL) K562 is a human erthroleukemia cell line, cultured in RPMI1640 (GIBCO/BRL) and 10% fetal bovine serum (Hyclone). C8166-45 is aIL-9 receptor bearing line, cultured in RPMI 1640 GIBCO/BRL) and 10%Fetal bovine serum (Hyclone). All the cell lines respond to cytokinesincluding IL-9. The cell lines are fed and reseeded at 2×10⁵ cells/mlevery 72 hours.

The cells were centrifuged for 10 minutes at 2000 rpm and resuspended inRPMI 1640 with 0.5% Bovine Serum Albumin (GIBCO/BRL, Gaithersburg, Md.)and insulin-transferrin-selenium (ITS) cofactors (GIBCO/BRL,Gaithersburg, Md.). Cells were counted using a hemocytometer and dilutedto a concentration of 1×10⁵ cells/ml and plated in a 96-well microtiterplate. Each well contained 0.15 or 0.2 ml giving a final concentrationof 2×10⁴ cells per well.

MO7e cells were stimulated with So ng/ml Stem Cell Factor (SCF) (R&DSystems, Minneapolis, Minn.) alone, 50 ng/ml SCF plus 50 ng/ml IL-3 (R&DSystems, Minneapolis, Minn.), or 50 ng/ml SCF plus 50 ng/ml IL-9. Acontrol was included which contains cells and basal media only. Serialdilutions of test compounds (i.e, recombinant IL-9 proteins, peptides,small molecules) were added to each test condition in triplicate. The MJcell line was used as an independent control for nonspecificcytotoxicity. Cultures were incubated for 72-96 hours at 37° C. in 5%CO₂.

Cell proliferation was assayed using the Abacus Cell Proliferation Kit(Clontech, Palo Alto, Calif.) which determines the amount ofintracellular acid phosphatase present as an indication of cell number.The substrate p-nitrophenyl phosphate (pNPP) was converted by acidphosphatase to p-nitrophenol which was measured as an indicator ofenzyme concentration. pNPP was added to each well and incubated at 37°C. for one hour. 1 N sodium hydroxide was then added to stop theenzymatic reaction, and the amount of p-nitrophenol was quantified usinga Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, Va.) at410 nm wavelength. Standard curves that compare cell number with opticalabsorbance were used to determine the linear range of the assay. Assayresults were only used when absorbance measurements are within thelinear range of the assay.

FIG. 14 illustrates the amino acid sequence of three peptide antagonistsof IL-9 function. Each peptide was incubated with MO7e cells andinhibition of cellular growth induced by IL-9 was determined bycomparison with control conditions (no peptide) (see FIGS. 15-17). Therewas no evidence for cytotoxicity with any of the peptides. PeptidesKP-16 and KP-20 are predicted to lie within two anti-parallelalpha-helicies and define a critical IL-9 receptor binding domain forthe IL-9 ligand. The protein polymorphism at codon 117 lies within KP-23and KP-24 which also exhibited antagonistic properties, furtherdemonstrating the importance of this region surrounding the site ofgenetic variation.

FIG. 18 illustrates the effect of tyrophostins (obtained fromCalbiochem) on the IL-9 dependent growth of MO7e cell in vitro. Eachtyrophostin was incubated with MO7e cells and inhibition of cellulargrowth induced by IL-9 was determined by comparison with controlconditions (no treatment). There was no evidence for cytotoxicity withany of the treatments. Tyrophostins B46 and B56 provided the greatestinhibition suggesting a common structure activity relationship.

FIG. 19 illustrates the effect of aminosterols isolated from the sharkliver as set forth in U.S. Ser. Nos. 08/290,826, 08/416,883, 08/478,763,and/or 08/483,059, incorporated herein by reference on the IL-9dependent growth of MO7e cell in vitro. Each aminosterol was incubatedwith MO7e cells at 20 μg/ml of the culture media and inhibition ofcellular growth induced by IL-9 was determined by comparison withcontrol conditions (no treatment). There was no evidence forcytotoxicity with any of the treatments. Aminosterols 3 and 6consistently provided the greatest inhibition of growth.

EXAMPLE 10 Assay for Proliferation of IgE Secreting Cells

B cell lines can be used to assess the function of rIL-9 and recombinantmutant forms of these proteins as well as other IL-9 antagonists. Theproliferation of IgE secreting cells is measured for rIL-9 and comparedto other cytokines or variant forms of rIL-9. In addition, compounds aretested for their ability to antagonize the baseline proliferativeresponse of IgE secreting cells to rIL-9. Once a baseline IgE responseis established for a cytokine, a statistically significant (P<0.05) lossof response in assays repeated three times in triplicate is consideredevidence for antagonism. A true antagonistic response is differentiatedfrom cellular toxicity by trypan blue staining (a technique well knownto one of normal skill in the art).

Cell Preparation and Cultures

Peripheral blood lymphocytes (PBL) are isolated from heparinized bloodof healthy donors or by mincing the spleens of mice. Mononuclear cellsare separated by centrifugation on Ficoll/Hypaque (Pharmacia, Uppsala,Sweden) gradients. Semi-purified human B lymphocytes are obtained byresetting with neuraminidase (Behring, Marburg, FRG)-treated sheep redblood cells and plastic adherence for 1 hour at 37° C. B cells are alsopurified using paramagnetic separation with anti-CD20 coated magneticbeads (DYNAL) according to the manufacturer's recommendations.

The relative proportion of B cells, T cells and monocytes is determinedby flow cytometry using monoclonal antibodies specific for CD23, CD3 andCD14, respectively (Becton Dickinson, Mountain View, Calif.). Briefly,10⁶ cells/ml are incubated with a 1:1000 dilution of phycoerythrinconjugated anti-CD23 and fluorescein-conjugated anti-CD3 and anti-CD14antibodies for 30 minutes at 4° C. After 3 spin washes with sterile PBSand 1% bovine serum albumin (Sigma) fluorescence is measured with acytofluorograph (FACSTAR Plus, Becton Dickinson, Grenoble, France).Typically, there are 45% CD20+, 35% CD3+ and 10% CD14+ cells in a countof 5000 cells per sample.

Cells are cultured at a density of 2×10⁶ cells/ml RPM1 1640 supplementedwith 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES (RPM1-FCS) at37° C. under a 5% CO2/95% air humidified atmosphere. Cultures areincubated with increasing concentrations of IL-4, rhIL-9, rmIL-9, orrecombinant mutant forms of these proteins, alone, or in combination.Competition experiments are run with mixtures of one or more of theserecombinant molecules or other IL-9 antagonists. The cultures aremaintained for 9-13 days.

Frequency of IgE-Secreting B Cells

The frequency of IgE-secreting human B cells in response to human ormurine IL-9 is determined using an ELISA-spot assay (Dugas et al., 1993,Eur J Immunol 23:1687-1692; Renz, H. et al., 1990. J. Immunology.145:3641. Nitrocellulose flat-bottomed 96-well plates are coatedovernight at 4° C. with purified goat antihuman IgE mAb diluted in 0.1 MNaHCO3 buffer (2.5 μg/ml). After one PBS-Tween 20 wash, plates areincubated for 1 hour with RPMI-FCS to saturate nonspecific bindingsites. B cells obtained after 9-13 days of culture are collected, washedthrice and resuspended at 10⁵ cells/ml RPMI-FCS, then transferred ontothe anti-IgE-coated plates followed by an 18 hour incubation at 37° C. Aperoxidase-conjugated mouse antihuman IgE mab at various dilutions isadded for 2 hours at 37° C. after washing. Spots are visualized afteraddition of diamino-benzidine diluted in 0.1 M Tris-HCl containing 0.03%H₂O₂. After 24 hours spots are counted with an inverted microscope at25× magnification. Data are expressed as the number of IgE-secretingcells per 10⁶ cells.

EXAMPLE 11 ELISA for IgE Secreted by Cells Co-Stimulated with IL-9

Cells are isolated, prepared and stimulated as described above inExample 10. Flat bottom microtiter plates (Nunc) are coated with rabbitanti-human IgE (1:2000, final dilution; Serotec, Oxford, GB), in 200 μlof 10 mM bicarbonate buffer (pH 9.6). After overnight incubation at 4°C., the plates are washed four times with phosphate-buffered saline(PBS) containing 0.05% Tween (PBS-Tween; Merck, Hohenbrunn, FRG) and areincubated for 1 h at room temperature with RPMI-FCS to saturatenonspecific protein-binding sites. After washing, 200 μl serialdilutions of human IgE (Eurobio, Les Ulis, France), standards inPBS-Tween are added to the respective plates to establish calibrationcurves. Dilutions of culture supernatants to be tested are then addedand, after 2 h at room temperature, the plates are washed and 200 μl ofdiluted specific alkaline phosphatase-conjugated anti-IgE (1:250;Serotec), anti-IgG or anti-IgM (Behring) is added in the appropriateplates. After 2 h at room temperature, the plates are washed and 200 μl(0.5 mg/ml) p-nitrophenylphosphate (Sigma) in citrate buffer is added.Plates are incubated at 37° C., and absorbance (A) is measured at 405 nmusing an autoreader (Dynatech Laboratories Inc, Chantilly, Va.). Thethreshold sensitivities of the assays are 100 pg/ml for IgE, 1 ng/ml forIgG, and 2 ng/ml for IgM and the variation between duplicatedeterminations of samples typically does not exceed 10%.

EXAMPLE 12 The Role of IL-9 in Murine Models of Asthma: The AirwayResponse of Unsensitized Animals

Animals

Certified virus-free male mice ranging in age from 5 to 6 weeks wereobtained from the Jackson Laboratory (Bar Harbor, Me.). Animals werehoused in high-efficiency particulate filtered air (HEPA) laminar flowhoods in a virus and antigen free facility and allowed free access topelleted rodent chow and water for 3 to 7 days prior to experimentalmanipulation. The animal facilities were maintained at 22° C. and thelight:dark cycle was automatically controlled (10:14 h light:dark). Maleand female DBA/2 (D2), C57BL/6 (B6), and (B6D2)F1 (F1) mice 5 to 6 weeksof age were purchased from the Jackson Laboratory, Bar Harbor, Me., orthe National Cancer Institute, Frederick, Md. BXD mice were purchasedfrom the Jackson Laboratory, Bar Harbor, Me. Food and water were presentad libitum.

Phenotyping and Efficacy of Pretreatment

To determine the bronchoconstrictor response, respiratory systempressure was measured at the trachea and recorded before and duringexposure to the drug. Mice were anesthetized and instrumented aspreviously described. (Levitt R C, and Mitzner W, FASEB J 2:2605-2608(1988); Levitt R C, and Mitzner W, J Appl Physiol 67(3): 1125-1132(1989); Kleeberger S, Bassett D, Jakab G J, and Levitt R C, Am J Physiol258(2)L313-320 (1990); Levitt R C; Pharmacogenetics 1:94-97 (1991);Levitt R C, and Ewart S L; Am J of Respir Crit Care Med151:1537-1542(1995); Ewart S, Levitt R C, and Mitzner W, In press, JAppl Phys (1995). Airway responsiveness was measured to one or more ofthe following: 5-hydroxytryptamine (5HT) (sigma). An additional branchconstruction that can be used is acetylcholine (sigma), atracurium(Glaxo welcome). A simple and repeatable measure of the change in P_(pi)following bronchoconstrictor challenge was used and which has beentermed the Airway Pressure Time Index (APTI) (Levitt R C, and Mitzner W,FASEB J 2:2605-2608 (1988); Levitt R C, and Mitzner W, J Appl Physiol67(3): 1125-1132 (1989). The APTI was assessed by the change in peakinspiratory pressure (P_(pi)) integrated from the time of injection tillthe peak pressure returned to baseline or plateaued. The APTI wascomparable to airway resistance (R_(rs)), however, the APTI includes anadditional component related to the recovery from bronchoconstriction.

The strain distribution of bronchial responsiveness was identified inmultiple inbred mouse strains in previous studies (Levitt R C, andMitzner W, FASEB J 2:2605-2608 (1988); Levitt R C, and Mitzner W, J ApplPhysiol 67(3): 1125-1132 (1989). The R_(rs) and/or APTI was determinedin A/J, C3H/HeJ, DBA/2J, C57BL/6J mice.

Prior to sacrifice whole blood was collected for serum IgE measurementsby needle puncture of the inferior vena cava in completely anesthetizedanimals. The samples were spun to separate cells and serum was collectedand used to measure total IgE levels. Samples not measured immediatelywere frozen at −20° C.

Bronchoalveolar lavage and cellular analyses was preformed as describedelsewhere (Kleeberger et al., 1990).

All IgE serum samples were measured using an ELISA antibody-sandwichassay. Microtiter plates (Corning #2585096, Corning, N.Y.) were coated,50 μl per well, with rat anti-mouse IgE antibody (Southern Biotechnology#1130-01, Birmingham, Ala.) at a concentration of 2.5 μg/ml in coatingbuffer of sodium carbonate-sodium bicarbonate with sodium azide (Sigma#S-7795, #S-6014 and #S-8032, St Louis, Mo.). Plates were covered withplastic wrap and incubated at 4° C. for 16 hours. The plates were washedthree times with a wash buffer of 0.05% Tween-20 (Sigma #P-7949) inphosphate-buffered saline (BioFluids #313, Rockville, Md.), incubatingfor five minutes for each wash. Blocking of nonspecific binding siteswas accomplished by adding 200 μl per well 5% bovine serum albumin(Sigma #A-7888) in PBS, covering with plastic wrap and incubating for 2hours at 37° C. After washing three times with wash buffer, duplicate 50μl test samples were added to the wells. Test samples were assayed afterbeing diluted 1:10, 1:50, and 1:100 with 5% BSA in wash buffer. Inaddition to the test samples a set of IgE standards (PharMingen #03121D,San Diego, Calif.) at concentrations from 0.8 ng/ml to 200 ng/ml in 5%BSA in wash buffer were assayed to generate a standard curve. A blank ofno sample or standard was used to zero the plate reader (background).After adding samples and standards, the plate was covered with plasticwrap and incubated for 2 hours at room temperature. After washing threetimes with wash buffer, 50 ul of second antibody rat anti-mouseIgE-horseradish peroxidase conjugate (PharMingen #02137E) was added at aconcentration of 250 ng/ml in 5% BSA in wash buffer. The plate wascovered with plastic wrap and incubated 2 hours at room temperature.After washing three times with wash buffer, 100 ul of the substrate 0.5mg/ml O-phenylaminediamine (Sigma #P-1526) in 0.1 M citrate buffer(Sigma #C-8532) was added to every well. After 5-10 minutes the reactionwas stopped with 50 μl of 12.5% H₂SO₄ (VWR #3370-4, Bridgeport, N.J.)and absorbance was measured at 490 nm on a Dynatech MR-5000 plate reader(Chantilly, Va.). A standard curve was constructed from the standard IgEconcentrations with antigen concentration on the x axis (log scale) andabsorbance on the y-axis (linear scale). The concentration of IgE in thesamples was interpolated from the standard curve.

EXAMPLE 13 The Role of IL-9 in Murine Models of Asthma: The AirwayResponse of Sensitized Animals

Animals, Phenotyping, and Optimization of Antigen Sensitization

Animals and handling were essentially as described in Example 12.Sensitization with turkey egg albumin (OVA) and aerosol challenge wascarried out to assess the effect on BHR, BAL, and serum IgE. OVA wasinjected I.P. (25 μg) day 0 prior to OVA or saline aerosolization. Micewere challenged with OVA or saline aerosolization which was given oncedaily for 5 to 7 days starting on either day 13 or 14. Phenotypicmeasurements of serum IgE, BAL, and BHR was carried out on day 21. Theeffect of a 7 day OVA aerosol exposure on bronchoconstrictor challengewith 5-HT and acetylcholine were evaluated along with serum total IgE,BAL total cell counts and differential cell counts, and bronchialresponsiveness. The effect of antibody (Ab) or saline pretreatment onsaline aerosol or OVA aerosol induced lung inflammation was examined bymeasuring BHR, BAL, and serum IgE. Ab were administered I.P. 2-3 daysprior to aerosolization of saline or OVA.

Lung histology was carried out after the lungs are removed during deepanesthesia. Since prior instrumentation may introduce artifact, separateanimals were used for these studies. Thus, a small group of animals wastreated in parallel exactly the same as the cohort undergoing variouspretreatments except these animals were not used for other tests asidefrom bronchial responsiveness testing. After bronchial responsivenesstesting, the lungs were removed and submersed in liquid nitrogen.Cryosectioning and histologic examination were carried out in a routinefashion.

Polyclonal neutralizing antibodies for murine IL-9 were purchased from R& D systems, Minneapolis, Minn. and blocking antibodies for murine IL-9receptor were produced for Magainin Pharmaceuticals Inc. by LampineBiological Labatories, Ottsville, Pa. using peptide conjugates producedat Magainin. The polyclonal antisera were prepared in rabbits againstpeptide sequences from the murine IL-9 receptor. The peptides used toproduce the antisera were: GGQKAGAFTC (residues 1-10) (SEQ ID NO:19);LSNSIYRIDCHWSAPELGQESR (residues 11-32) (SEQ ID NO:20); andCESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue added to theN-terminus for coupling the peptide to the carrier protein) (SEQ IDNO:21). The antisera were generated using techniques described inProtocols in Immunology, Chapter 9, Wiley. Briefly, the peptides werecoupled to the carrier protein, Keyhole Limpet, hemocyanin, (Sigma)through the side chain of the Cys residue using the bifunctionalcross-linking agent MBS (Pierce). Peptide conjugates were used toimmunize rabbits with appropriate adjuvents and useful antisera wasobtained after several booster injections of the peptide conjugate. Theantibodies were used therapeutically to down regulate the functions ofIL-9 and assess the importance of this pathway to baseline lungresponsiveness, serum IgE, and BAL in the unsensitized mouse. After Abpretreatment on baseline BHR, BAL, and serum IgE levels relative tocontrols was determined. In additional experiments, recombinant humanand murine IL-9 were administered I.P. 1 day before and daily duringantigen sensitization (days 13-18). The animals were then phenotyped asdescribed.

The phenotypic response of a representative animal treated with salineI.P. on day zero and challenged on days 14-20 with saline (as describedin Example 12) is shown in FIG. 20 panel 1 (top). Baseline (control)serum total IgE was 9.2 ng/ml. Bronchoalveolar lavage (BAL) total cellcounts showed 182,500 cells per milliliter of BAL. These animals did notdemonstrate bronchial hyperresponsiveness when compared to historicalcontrols (Levitt R C, and Mitzner W, J Appl Physiol 67(3): 1125-1132;1989).

FIG. 20 panel 2 (top middle) shows a representative animal from a grouppresensitized with OVA I.P on day zero and challenged with saline ondays 14-20. These animals did not differ in their response tobronchoconstrictor, serum IgE, or BAL cell counts from the unsensitizedmice (FIG. 7 top panel).

FIG. 20 panel 3 (bottom middle) shows a representative animal from thosepresensitized with OVA I.P on day zero and challenged with antigen (OVA)on days 14-20. These animals developed bronchial hyperresponsiveness(approximately two to three-fold over controls), elevated serum IgE(nearly one thousand-fold over controls), and increased numbers ofinflammatory cells in the airway as demonstrated by elevated BAL cellcounts (approximately thirty-fold) as compared to controls (FIG. 20 top2 panels). Most of the cells recruited to the airway as a result of thisantigen challenge were eosinophils.

FIG. 20 panel 4 (bottom) shows a representative animal from thosepresensitized with OVA I.P on day zero, pretreated with polyclonalneutralizing antibodies for murine IL-9 (approximately 200 Ag/mouse I.P.in 0.5 ml of PBS), and challenged with antigen (OVA) on days 14-20.These animals were protected from the response to antigen. They did notdiffer significantly in their bronchial responsiveness, serum IgE, orBAL cell counts from controls (FIG. 20 top 2 panels).

FIG. 21 illustrates the effect of antigen challenge to OVA (as describedabove) with and without pretreatment with polyclonal neutralizingantibodies to murine IL-9 I.P. three days prior in representativeanimals. The left figure (A1-2-1B) is a histologic section from thelungs of control animals (sensitized to OVA but exposed only to a salineaerosol challenge). The middle figure (A1-3-5) is a histologic sectionfrom the lungs of animals sensitized to OVA and exposed to an OVAaerosol challenge. The right figure (A1-4-5) is a histologic sectionfrom the lungs of animals sensitized to OVA and exposed to an OVAaerosol challenge who were pretreated three days prior with polyclonalneutralizing antibodies to murine IL-9. Pretreatment with neutralizingantibody produced histological confirmation of complete protection fromantigen challenge.

FIG. 22 panel 1 (top) shows a representative animal from micepresensitized with OVA I.P on day zero and challenged with antigen (OVA)on days 13-18. These animals developed bronchial hyperresponsiveness(approximately two to three-fold over controls), and increased numbersof inflammatory cells including eosinphils in the airways asdemonstrated by elevated BAL cell counts as compared to controls (FIG.20 top 2 panels). Many of the cells recruited to the airway as a resultof this antigen challenge were eosinophils.

FIG. 22 panel 2 (bottom) shows a representative animal from thosepresensitized with OVA I.P on day zero, pretreated with polyclonalneutralizing antibodies to the murine IL-9 receptor (approximately 1mg/mouse I.P. in 0.5 ml of PBS), and challenged with antigen (OVA) ondays 13-18. This representative animal was protected from the responseto antigen. This response did not differ significantly bronchialresponsiveness, BAL cell counts from controls (FIG. 20 top 2 panels).These data demonstrate the potential effectiveness of treating atopicallergy with antibodies to the IL-9 receptor.

EXAMPLE 14 Murine Spleen Isolation and Culture

Mice were anesthetized and spleens were removed aseptically. Spleenswere minced with scissors and gently passed through a wire mesh(autoclaved) [#60 sieve]. Cells were resuspended in 40 mls of RPMI-1640[GIBCO, BRL, Rockville, Md.), and spun for 5 min. at 250×G twice. Thepellet was resuspended in 10 mls of lysing butter to remove RBCs [4.15gm NH4Cl, 0.5 gm KHCO3; 019 g EDTA to 500 mls with ddH20]. Cells wereincubated for about 5 minutes at 37° C. and 40 mls of RPMI-FCS[RPMI-1640, 10% AFBS, 50 μM BME 2 mM glutamine, containing penicillinand streptomycin]. These cells were spun again for 5 minutes at 250×Gand resuspended in 20 mls RPMI-FCS with or without 5 μg/ml ofconcanavalin A [Sigma #C5275]. IL-9 was assessed at 48 hours inuntreated splenocytes and after concanavalin A stimulation from DBA/2J(D2) and C57BL/6J (B6) mice. IL-9 was amplified by RT-PCR (as set forthin Example 6), and probed with an IL-9 specific murine probe afterSouthern transfer. Southern blots were performed by “standard”techniques. Briefly, RT-PCR products were electrophoresed in 2% agarosegels. Gels were stained with ethidium bromide and photographed with aruler to determine molecular weight of DNA in southern blot. Gels werethen soaked in 0.5N NaOH for 30 minutes and neutralized in 0.5M Tris, pH7.0 for 30 minutes. DNA was transferred to zetaprobe (BioRAD) nylonmembrane by capillary transfer in 20×SSC overnight. The next day, themembrane was air dried, baked at 80° C. for 15 minutes and prehybridizedin 6×SSC and 0.1% SDS for 1 hour at 42° C. A kinase end-labelled p32oligonucleotide probe (5′-AATTACCTTATTGAAAATCTGAAG-3′) was added to thehybridization solution plus 0.1 mg/ml sheared salmon sperm DNA andincubated overnight at 42° C. The next day, the filter was washed in3×SSC and 0.1% SDS at 37° C. for 30 minutes, and the filter was exposedto film for 1 hour. FIG. 26 illustrates steady state levels of IL-9after 48 hours from each strain of mice. IL-9 was observed inunstimulated D2 (D2−) splenocytes, whereas no IL-9 was detectable in B6(B6−) mice. While there was a significant increase of IL-9 afterconcanavalin A stimulation in D2 (D2+) splenocytes, there was nodetectable It-9 in B6 (B6+) mice despite concanavalin A treatment.

EXAMPLE 15 Expression of Human Met117 IL-9 and Thr117 IL-9 in PBMCs

SDS-PAGE and Immunoblot Analysis

After obtaining proteins isolated from human PBMC of healthy donorsinhibiting either the wild type (Thr117) or Met117-IL-9 genotypes as setforth in Example 13, and SDS-PAGE was performed by the method of Laemmli(Laemmli U.K. (1970) Nature 227, 680-685) by using a 18% polyacrylamidegel in a mini-gel system (Xcell II vertical gel unit, Novex). Forimmunoblot analysis, the proteins separated by SDS-PAGE were transferredto nitrocellulose membranes by using the SD transblot transfer unit(Biorad) in 25 mM Tris-glycine buffer, pH 8.3, containing 15% methanol(Towbin H., et al., (1979) (Proc. Natl. Acad. Sci. U.S.A. 76,4350-4354). The unoccupied binding cites on the membrane were blocked byincubating for 1 hour to overnight with 20 mM Tris-HCl buffer, pH 8.0,plus 0.05% tween 20 (TBST) containing 5% dry milk. The membranes werethen incubated with 1:1000 dilution of goat anti-human IL-9 polyclonalantibody (R&D Systems) for 1 hour at room temperature. The membraneswere washed with TBST and treated with 1:10,000 dilution of mouseanti-goat TgG conjugated with horseradish peroxidase for 1 hour. Afterwashing with TBST, the bound antibodies were visualized by addition ofthe super signal substrate chemiluminescence system (Pierce).

FIG. 24 demonstrates the expression of human IL-9 proteins from culturedPBMCs 48 hours after mitogen stimulation in individuals whose genotypeshave been determined by genomic analysis of the IL-9 gene. Lane 1 ismolecular weight markers, lane 2 is a Met117 homozygote, lane 3 is aheterozygote Met117/Thr117, lane four is a Thr117 homozygote. A singleproduct of the approximate expected size (14 kD) was seen in eachindividual PBMCs after mitogen stimulation. These data demonstrate thatboth forms of the IL-9 protein are expressed and stable at steady state.

While the invention has been described and illustrated herein byreferences to various specific materials, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

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Other embodiments of the invention described above and will be apparentto those skilled in the art from consideration of the specification andpractice of the invention disclosed within. It is intended that thespecification and examples considered as exemplary only, with true scopeand spirit of the invention being indicated by the following claims:

1-99. (canceled)
 100. A composition comprising an IL-9 antagonistselected from the group consisting of one or more peptides comprising anamino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.101-109. (canceled)
 110. The composition of claim 100, wherein thepeptide comprises the amino acid sequence of SEQ ID NO:
 13. 111. Thecomposition of claim 100, wherein the peptide comprises the amino acidsequence of SEQ ID NO:
 14. 112. The composition of claim 100, whereinthe peptide comprises the amino acid sequence of SEQ ID NO:
 15. 113. Amethod of treating a disease associated with an IL-9 over-expressioncomprising administration of a composition comprising one or morepeptides comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15.114. The method of claim 113, wherein the peptide comprises the aminoacid sequence of SEQ ID NO:
 13. 115. The method of claim 113, whereinthe peptide comprises the amino acid sequence of SEQ ID NO:
 14. 16. Themethod of claim 113, wherein the peptide comprises the amino acidsequence of SEQ ID NO: 15.